Construct capable of release in closed circular form from a larger nucleotide sequence permitting site specific expression and /or developmentally regulated expression of selected genetic sequences

ABSTRACT

The present invention relates generally to constructs and in particular genetic constructs comprising polynucleotide sequences capable of release in covalently closed, circular form from a larger nucleotide sequence such as[, but not limited to,] a genome of a eukaryotic cell. Preferably, once released, a polynucleotide sequence is reconstituted in a form which permits expression of the polynucleotide sequence. In one embodiment, the reconstituted polynucleotide sequence comprises a coding sequence with all or part of an extraneous nucleotide such as[, but not limited to,] an intronic sequence or other splice signal inserted therein. Expression and in particular transcription of the coding sequence involves splicing out the extraneous sequence. The release and circularization is generally in response to a stimulus such as a protein-mediated stimulus. More particularly, the protein is a viral or prokaryotic or eukaryotic derived protein or developmentally and/or tissue specific regulated protein.

FIELD OF THE INVENTION

[0001] The present invention relates generally to constructs and inparticular genetic constructs comprising polynucleotide sequencescapable of release in covalently closed, circular form from a largernucleotide sequence such as, but not limited to, a genome of aeukaryotic cell. Preferably, once released, a polynucleotide sequence isreconstituted in a form which permits expression of the polynucleotidesequence. In one embodiment, the reconstituted polynucleotide sequencecomprises a coding sequence with all or part of an extraneous nucleotidesuch as, but not limited to, intronic sequence or other splice signalinserted therein. Expression and in particular transcription of thecoding sequence involves splicing out the extraneous sequence. Accordingto this embodiment, the coding sequence may encode a peptide,polypeptide or protein, an antisense or sense nucleic acid molecule or aribozyme. In another embodiment, the reconstituted polynucleotidesequence will comprise the extraneous polynucleotide sequence locatedbetween a promoter element and coding sequence. In this embodiment,expression of the coding sequence is not substantially adverselyaffected by the presence of the extraneous sequence. In still a furtherembodiment, the reconstituted sequence forms an RNA promoter or otherregulatory genetic sequence comprising the extraneous sequence whichdoes not affect the function of the promoter. The release andcircularization is generally in response to a stimulus such as aprotein-mediated stimulus. More particularly, the protein is a viral orprokaryotic or eukaryotic derived protein or developmentally and/ortissue specific regulated protein. The construct of the presentinvention is particularly useful in conferring genetic resistanceagainst pathogens or inducing apoptosis or other cell death mechanismsuseful, for example, in treating cancer or inducing male or femalesterility in plants. The constructs permit, therefore, site specificexpression and/or developmentally regulated expression of selectedgenetic sequences.

BACKGROUND OF THE INVENTION

[0002] Reference to any prior art in this specification is not, andshould not be taken as, an acknowledgment or any form of suggestion thatthis prior art forms part of the common general knowledge in Australiaor any other country.

[0003] The increasing sophistication of recombinant DNA technology isgreatly facilitating research and development in a range oftechnological fields such as the medical, horticultural and agriculturalindustries. Of particular importance, is the exploitation of naturallyoccurring genetic mechanisms in, for example, pathogens, to induceuseful phenotypic changes in cells, such as plant cells. This isparticularly evident in the horticultural field in relation tomechanisms to induce resistance in plants to a range of plant pathogens.

[0004] There has been considerable progress in the development of virusresistance in crops, for example, through transformation with transgenesderived from the target viruses. The most successful use of transgeneshas been with RNA plant viruses. However, despite a number attempts tocontrol single stranded DNA (ssDNA) plant viruses through transgenicresistance, strategies which have been successful for RNA plant viruseshave not been as effective for ssDNA plant viruses. DNA plant virusesand particularly the single stranded DNA (ssDNA) plant viruses areresponsible for significant commercial losses in a wide range of fruit,vegetable, grain and fibre crops in the tropics and sub-tropics.

[0005] There have been two groups of ssDNA viruses which infect plants.These are the Geminiviridae and the recently described nanovirus group.Members of the Geminiviridae have geminate virions and either amonopartite or bipartite circular ssDNA genome. Each molecule is about2.7 kb in length. Of the Geminiviridae genera, the begomoviruses and themastreviruses are the most important. The begomoviruses arewhitefly-transmitted and have either monopartite or bipartite genomes.Members of this genus include some of the most economically devastatingviruses of modern agriculture such as tomato (yellow) leaf curl(consisting of a range of different viruses spread through most tropicaland sub-tropical regions), African cassava mosaic (Africa), bean goldenmosaic (South and Central America), mungbean yellow mosaic (India) andcotton leaf curl (South and South-East Asia) viruses. The impact of manyof the begomoviruses has increased dramatically over recent years as aresult of the widespread introduction of the aggressive “B biotype” ofthe whitefly vector, Bemesia tabaci. The mastreviruses have had a lesserimpact on agriculture but are responsible for significant losses in somecrops. These viruses are transmitted by the leafhoppers and havemonopartite genomes. Members of this genus include maize streak(Africa), wheat dwarf (Europe) and tobacco yellow dwarf (Australia)viruses.

[0006] The nanoviruses have isometric virions and circular ssDNA genomesbut these genomes are multi-component with at least six differentintegral genomic components each of which is approximately 1 kb. Theseviruses are transmitted by aphids except for one tentative nanovirus,coconut foliar decay virus, which is transmitted by a treehopper and hasonly been reported from Vanuatu. The economically most importantnanovirus is banana bunchy top virus (BBTV) which nearly destroyed theAustralian banana industry in the 1920s and causes major losses in theSouth Pacific, Asia and Africa. Subterranean clover stunt (Australia),faba bean necrotic yellows (Mediterranean) and coconut foliar decay(Vanuatu) viruses all cause significant yield loss.

[0007] The genome organization of the begomoviruses, the mastrevirusesand the nanoviruses have significant differences including the numberand size of genomic components and number and size of genes, theprocessing of transcripts, the orientation of genes and the like. Thereare, however, remarkable similarities which suggest that these viruseshave very similar replication and infection strategies. All the gemini-and nanoviruses encode (i) a Rep protein which has nicking and joiningactivity and directs rolling circle replication of the viral genome;(ii) a virion coat protein; (iii) a protein that is involved in bindinghost cell retinoblastoma-like proteins resulting in the cell moving to Sphase; (iv) a cell-to-cell movement protein; and (v) a nuclear shuttleprotein. Further, the viruses have functionally similar intergenicregions (IR). For instance, the IR of begomoviruses, the LIR ofmastreviruses and the CR-SL of banana bunchy top nanovirus all contain(i) a stem/loop structure, the nonanucleotide loop sequence of which ishighly conserved between all gemini- and nanoviruses and is the site ofnicking and ligation by the Rep protein; and (ii) a domain within thisregion which recognizes the Rep protein. The SIR of the mastrevirusesand the CR-M of banana bunchy top nanovirus are the site of binding ofan endogenous primer responsible for priming the conversion of virionssDNA into transcriptionally active dsDNA.

[0008] The success in developing transgenic resistance to RNA viruses incrops and the increasing demand for such resistance to ssDNA viruses hasresulted in investigation of a wide range of strategies for ssDNAviruses targeting various viral genes including the coat protein gene,movement protein gene and the Rep protein gene. In addition, strategiesusing defective interfering DNAs and a suicide gene have beeninvestigated. Most work in this area has involved begomoviruses ratherthan mastre- or nanoviruses.

[0009] In work leading up to the present invention, the inventors haveexploited the replication mechanisms of ssDNA viruses in order to inducegenetic resistance in plants. However, the present invention has wideranging applications in modulating genetic activities such as expressionof polynucleotide sequences to effect a particular phenotype in responseto a stimulus.

SUMMARY OF THE INVENTION

[0010] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

[0011] Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1, <400>2, etc. A sequence listing isprovided after the claims.

[0012] One aspect of the present invention provides a constructcomprising a genetic element operably flanked by nucleotide sequencesrecognizable by a viral-derived, replication-facilitating protein or itsderivatives or eukaryotic and prokaryotic cell homologues whenintegrated into the genome of a eukaryotic cell which viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic cell homologues facilitates excision and circularization ofthe genetic element and all or part of the flanking nucleotide sequencesand wherein said nucleotide sequences recognizable by saidviral-derived, replication-facilitating protein or its derivatives oreukaryotic or prokaryotic cell homologues are adjacent to or insertedwithin one or more extraneous sequences including intron sequences orparts thereof or other splice signals wherein the genetic element andother nucleotide sequences, in a non-circular form, comprise two modularnucleotide sequences which, upon circularization, form a geneticsequence exhibiting a property or a capacity for exhibiting a propertyabsent in the two modular nucleotide sequences prior to circularizationor prior to circularization and expression.

[0013] Another aspect of the present invention provides a constructcomprising a genetic element flanked by Rep-protein recognitionsequences or functional homologues from other viruses or eukaryotic orprokaryotic cells which facilitate the generation of a circularnucleotide sequence comprising said genetic element in the presence of aRep protein or its functional derivatives or homologues wherein saidRep-protein recognition sequences are adjacent to or inserted within oneor more recognition sequences, said genetic element comprising apolynucleotide sequence operably linked to regulatory sequences requiredto permit expression of said polynucleotide sequence when said geneticelement is contained within a circularized molecule wherein the geneticelement in linear form comprises in the 5′ to 3′ order:—

[0014] a polynucleotide sequence; and

[0015] regulatory sequences to permit expression of said polynucleotidesequence when in circular form,

[0016] such that upon circularization the genetic element comprises theregulatory sequence separated from the polynucleotide sequence by all orpart of a Rep protein-recognition sequence wherein upon expression, saidpolynucleotide sequence encodes an expression product.

[0017] A further aspect of the present invention provides a constructcomprising in 5′ to 3′ order first, second, third, fourth, fifth andsixth nucleotide sequences wherein:

[0018] the first and sixth nucleotide sequences may be the same ordifferent and each comprises a Rep protein-recognition sequence capableof being recognized by one or more Rep proteins or derivatives orhomologues thereof such that genetic material flanked by said first andsixth sequences including all or part of said first and sixth sequenceswhen said construct is integrated in a larger nucleotide sequence suchas a genomic sequence, is capable of being excised and circularizedwherein said Rep-protein recognition sequences are adjacent to orinserted within one or more extraneous sequences including intronicsequences or parts thereof or other splice signals;

[0019] the second nucleotide sequence comprises a 3′ portion ofpolynucleotide sequence;

[0020] the third nucleotide sequence is a transcription terminator orfunctional derivative or homologue thereof operably linked to saidsecond sequence;

[0021] the fourth nucleotide sequence is a promoter sequence operablylinked to the fifth nucleotide sequence; and

[0022] the fifth nucleotide sequence is a 5′ portion of a polynucleotidesequence wherein the 5′ and 3′ portions of said polynucleotide sequencerepresent a full coding sequence of said polynucleotide sequence;

[0023] wherein in the presence of one or more Rep proteins, when theconstruct is integrated into a larger nucleotide sequence such as agenomic sequence, a circularized genetic sequence is generated separatefrom said larger nucleotide sequence comprising in order said promotersequence operably linked to a polynucleotide sequence comprising all orpart of the extraneous sequence or other splice signal comprising all orpart of said first and/or sixth nucleotide sequences and a transcriptionterminator sequence.

[0024] Still another aspect of the present invention is directed to agenetic element for use in generating a construct, said genetic elementcomprising in 5′ to 3′ direction, a 3′ portion of a polynucleotidesequence operably linked to a transcription terminator; a promoteroperably linked to a 5′ portion of a polynucleotide sequence whereinupon circularization, the 5′ portion of the polynucleotide sequence isoperably linked to said 3′ portion of the polynucleotide sequenceseparated by all or part of an extraneous sequence or intron sequence orother splice signal.

[0025] Yet another aspect of the present invention provides a constructcomprising the nucleotide sequence substantially as set forth in SEQ IDNO:31 to SEQ ID NO:36 or a nucleotide sequence having 60% similarity toeach of SEQ ID NO:31 to SEQ ID NO:36 or a nucleotide sequence capable ofhybridizing to one or more of SEQ ID NO:31 to SEQ ID NO:36 or acomplementary form thereof under low stringency conditions at 42° C.

[0026] Even yet another aspect of the present invention contemplates amethod for generating a transgenic plant or progeny thereof resistant toa ssDNA virus, said method comprising introducing into the genome ofsaid plant a construct comprising in the 5′ to 3′ order, a Repprotein-recognition sequence adjacent to or within an intronic sequenceor other splice signal, a 3′ end portion of a polynucleotide sequence, atranscription terminator or its functional equivalent, a promotersequence operably linked to a 5′ end portion of the polynucleotidesequence wherein the 5′ and 3′ portions of the polynucleotide sequencerepresent the coding region of a peptide, polypeptide or protein capableof inducing cell death or dormancy, and same or different Repprotein-recognition sequences; wherein upon infection of said plantcells by ssDNA virus having a Rep protein which is capable ofrecognizing the flanking Rep protein-recognition sequences, theconstruct is excised and circularizes thus reconstituting saidpolynucleotide sequence in a form which is expressed into a peptide,polypeptide or protein which kills the plant cell or otherwise rendersthe plant cell dormant.

[0027] Even still another aspect of the present invention provides aconstruct comprising a genetic element flanked by a Repprotein-recognition sequences which facilitate the generation of acircular nucleotide sequence comprising said genetic element in thepresence of a Rep protein or its functional derivatives or homologueswherein said Rep-protein recognition sequences are adjacent to orinserted within one or more extraneous sequences including intronicsequences or parts thereof or other splice signal, said genetic elementcomprising a 3′ portion and a 5′ portion of a promoter separated by alength of a nucleotide sequence to substantially prevent functioning ofsaid promoter, said genetic element in linear form comprises in the 5′to 3′ order:—

[0028] a 3′ portion of said promoter;

[0029] optionally a polynucleotide sequence operably linked to said 3′portion of said promoter; and

[0030] a 5′ portion of said promoter, such that upon circularization thegenetic element comprises the 5′ and 3′ portions of the promotersequence separated by all or part of a Rep protein-recognition sequenceand/or intron sequences or other splice signal but which does notinactivate the activity of the promoter, said circular moleculeoptionally further comprising the promoter operably linked topolynucleotide sequence.

[0031] The promoter may be a DNA promoter or an RNA promoter.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIG. 1 is a diagrammatic representation of pTBN.

[0033]FIG. 2 is a diagrammatic representation of pTBN6.

[0034]FIG. 3 is a diagrammatic representation of pTBN1.

[0035]FIG. 4 is a diagrammatic representation of pRTBN6.

[0036]FIG. 5 is a diagrammatic representation of pRTBN1.

[0037]FIG. 6 is a diagrammatic representation of pRTBN 1/6.

[0038]FIG. 7 is a schematic representation of plasmid p35S-2301.

[0039]FIG. 8 is a schematic representation of plasmid pTEST1.

[0040]FIG. 9 is a schematic representation of plasmid pTEST2.

[0041]FIG. 10 is a schematic representation of plasmid pTEST3.

[0042]FIG. 11 is schematic representation of plasmid pTEST4.

[0043]FIG. 12 is a schematic representation of plasmid pBI-TYDV1.1 mer.

[0044]FIG. 13 is a schematic representation of plasmid p35S-Rep.

[0045]FIG. 14 is a graphical representation of the results of a celldeath assay using expression vectors. Error bars show 95% confidenceintervals.

[0046]FIG. 15 is a graphical representation of the results ofrecircularization cell death assay using expression vectors. Error barsshow 95% confidence intervals.

[0047]FIG. 16 is a graphical representation of the results from theinducible recircularization cell death assay using expression vectors.Error bars show 95% confidence intervals.

[0048]FIG. 17 is a schematic representation of plasmids (A) p35S-BTR-LIRand (B) p35S-BUTR-LIR.

[0049]FIG. 18 is a schematic representation of plasmids (A) p35S-BTR and(B) p35S-BUTR.

[0050]FIG. 19 is a schematic representation of plasmids (A) pBTR.test1and (B) pBUTRtest1.

[0051]FIG. 20 is a diagrammatic representation of pGI.

[0052]FIG. 21 is a diagrammatic representation of pGI6.

[0053]FIG. 22 is a diagrammatic representation of pGI1.

[0054]FIG. 23 is a diagrammatic representation of pRGI6.

[0055]FIG. 24 is a diagrammatic representation of pRGI1.

[0056]FIG. 25 is a diagrammatic representation of pRGI 1/6.

[0057]FIG. 26 is a schematic representation of a proposed model forRep-activated expression of human serum albumin from plasmid pHSA1.

[0058]FIG. 27 is a diagrammatic representation of a construct for use insense/antisense modulation of genetic expression.

[0059] A summary of sequence identifiers is provided herewith.

SUMMARY OF SEQENCE IDENTIFIERS

[0060] SEQUENCE IDENTIFIER DESCRIPTION SEQ ID NO: 1 to SEQ ID NO: 18Synthetic oligonucleotide SEQ ID NO: 19 to SEQ ID NO: 44 Primers SEQ IDNO: 45 to SEQ ID NO: 52 Synthetic oligonucleotide SEQ ID NO: 66 BarnasepTBN SEQ ID NO: 67 Barnase pRTBN6 SEQ ID NO: 68 Barnase pRTBN1 SEQ IDNO: 69 GFP pGI SEQ ID NO: 70 GFP pGI6 SEQ ID NO: 71 GFP pGI1 SEQ ID NO:72 primer SEQ ID NO: 73 primer

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present invention is predicated in part on the recognitionthat a viral-derived, replication-facilitating protein may be used toexcise and circularize specific, targeted sequences from the genome of aeukaryotic cell. The term “excise” includes in this case release andmore particularly replicative release of targeted sequences. Theviral-derived, replication-facilitating proteins initiate nicking,excision and circularization of genetic elements flanked by particularsequences specific for and recognized by the viral-derived,replication-facilitating protein. The present invention extends toderivatives of the viral-derived facilitating proteins and eukaryoticand prokaryotic homologues thereof The present invention extends to,therefore, any sequences capable of facilitating cleavage and ligationof polynucleotide sequences and are referred to hereinafter as“recognition sequences” and may also be considered herein as “extraneoussequences”. The recognition sequences are adjacent to or inserted withinextraneous sequences including intronic sequences or other splicesignals.

[0062] In one embodiment, the circularization process permits theformation of a particular genetic sequence from two modular componentsseparated by a splicable extraneous sequence upon expression. Prior tocircularization, the modular components are genetically separated andhence not operably linked. Operable linkage is conveniently shown, forexample, in one embodiment, by the ability for the genetic sequencecomprising the modular components to be expressed. The term “expressed”in this instance includes transcription to an mRNA sequence andoptionally translation of the mRNA sequence to a translation product.Expression, however, is not the sole criterion for constitution of agenetic sequence from modular components. In another embodiment, thegenetic sequence produced following circularization may have otheruseful functions not requiring expression. For example, the resultinggenetic sequence may comprise a protein binding recognition sequencethereby targeting particular cytoplasmic or nuclear proteins.Alternatively, the reconstituted polynucleotide sequence comprises anextraneous sequence between the promotors elements and a codingsequence. In the case of the former, the promoter is preferably an RNApromoter such as from a TMV, AMV or TEV virus. Alternatively, thepromoter is a DNA promoter where the insertion of the recognition doesnot substantially adversely affect its activity.

[0063] Accordingly, one aspect of the present invention provides aconstruct comprising a genetic element operably flanked by nucleotidesequences recognizable by a viral-derived, replication-facilitatingprotein or its derivatives or eukaryotic and prokaryotic cell homologueswhen integrated into the genome of a eukaryotic cell whichviral-derived, replication-facilitating protein or its derivatives oreukaryotic or prokaryotic cell homologues facilitates excision andcircularization of the genetic element and all or part of the flankingnucleotide sequences and wherein said nucleotide sequences recognizableby said viral-derived, replication-facilitating protein or itsderivatives or eukaryotic or prokaryotic cell homologues are adjacent toor inserted within one or more extraneous sequences including intronsequences or parts thereof or other splice signals wherein the geneticelement and other nucleotide sequences, in a non-circular form, comprisetwo modular nucleotide sequences which, upon circularization, form agenetic sequence exhibiting a property or a capacity for exhibiting aproperty absent in the two modular nucleotide sequences prior tocircularization or prior to circularization and expression.

[0064] The term “construct” is used in its broadest sense and includes agenetic construct, nucleic acid molecule, vector, plasmid or any othernucleotide sequence comprising at least two heterologous sequences. Theconstruct, therefore, is a recombinant molecule engineered to comprisetwo or more nucleotide sequences from different genetic sources. In oneembodiment, the construct is in an isolated form. The term “isolated”includes biologically pure, substantially pure or in another conditionwhere at least one purification step has been performed on a samplecomprising the construct. A “purification step” includes, for example, aprecipitation, centrifugation and/or a chromatographic orelectrophoretic separation. In another embodiment, the genetic constructis integrated into the genome of a host cell. The construct may comprisenucleotide sequences which are lost, removed or rearranged followingintegration. In yet another embodiment, the construct is in circularform either generated in vitro or following excision from the genome ofthe host cell.

[0065] The term “genetic element” is used in its broadest sense andincludes a series of two or more nucleotide sequences engineered in aparticular order relative to the 5′ to 3′ or 3′ to 5′ orientations ofthe genetic element. In essence, the genetic element comprises twonucleotide sequences in modular form. The term “modular” is not toimpart any limitation to the construction or structure of the nucleotidesequences but emphasizes that a single genetic sequence is divided intotwo components, i.e. modular components. Upon circularization, the twomodular components are orientated together to constitute, after removalof any extraneous sequences including intronic and splice sequences orother recognition sequences, a single genetic sequence exhibiting aparticular activity or property not present when the genetic sequence isin separate modular form. In certain circumstances, extraneous sequencesintervening the two modular components when in circular form may notneed to be removed if their presence does not substantially adverselyaffect the function of the modular components when constituted in thecorrect orientation relative to each other after circularization.Generally, the modular components are referred to as 5′ portions and 3′portions of a polynucleotide sequence. A portion comprises from a fewnucleotides (i.e. from about 2 to about 500) to many (i.e. from about501 to about 10,000). The 5′ and 3′ portions may encompass a centralportion.

[0066] In a preferred embodiment, the genetic element comprises, when incircular form, a promoter operably linked to the genetic sequencecomprising the two modular sequences. The two modular sequences may beseparated by an intronic sequence, splice signal or other recognitionsequence. The genetic element comprises, therefore, in linear form inthe 5′ to 3′ direction, a first modular nucleotide sequence comprisingthe 3′ portion of a polynucleotide sequence, a promoter sequenceoperably linked to the 5′ portion of the above-mentioned polynucleotidesequence. Upon circularization, the 3′ portion of the polynucleotidesequence is now orientated and fused by base linkage to the 5′ portionof the polynucleotide sequence thus reconstituting a functionalpolynucleotide sequence operably linked to a promoter. Depending on theconstruct, an intronic sequence, splice signal or other recognitionsequence may separate the 5′ and 3′ portions of the reconstitutedpolynucleotide sequence. Upon processing during expression, the intronicsequence, splice signal or other recognition sequence may be excised. Ina particularly preferred embodiment, the genetic element comprises atranscription terminator sequence operably linked and downstream of the3′ portion of the polynucleotide sequence. Terms such as “promoter” and“terminator” are used in their broadest sense and are described in moredetail below.

[0067] In another embodiment, the reconstituted polynucleotide sequenceencodes an intronic, splice signal or other recognition sequence locatedbetween the promoter element and the coding sequence. According to thisembodiment, the recognition sequence would not be removed duringtranscription.

[0068] In yet another alternative embodiment, the genetic elementcomprises two modular components of a promoter or other regulatorysequence. Preferably, the modular components form a promoter sequenceafter circularization. If an intronic sequence, splice signal or otherrecognition sequence separates the modular components of a promotersequence, then such a sequence does not destroy or partially destroy theactivity of the promoter sequence. Alternatively, the promoter is an RNApromoter such as a promoter from TMV, AMV or TEV.

[0069] The polynucleotide sequence, when reconstituted, exhibits anactivity or property or a capacity to exhibit an activity or propertynot present in the separate modular nucleotide sequences prior to fusionfollowing circularization. Such an activity or property includes theability to encode a peptide, polypeptide or protein having a particularfunction, the ability to encode a mRNA sequence which may subsequentlybe translated into a peptide, polypeptide or protein or which may act asan antisense or sense molecule for down-regulation of a host gene orother genetic sequence or acting as a promoter or other regulatorysequence. Another property contemplated by the genetic sequencesincludes the ability to bind to protein to interact with nucleicregulatory sequences or to act or encode ribozyme and/or deoxyribozymemolecules.

[0070] Of particular importance, the genetic sequence may encodeproteins having enzymic activity, regulatory activity or structuralactivity or exhibit a therapeutic activity if administered to a mammalsuch as a human or livestock animal. Examples of the latter type ofmolecule include cytokines, interferons and growth factors. Proteinshaving enzymic activity are particularly preferred and such proteins areuseful in activating a biochemical pathway, facilitating the flow ofmetabolites down a particular pathway, conferring a property such asresistance to an insecticide, fungicide or herbicide or conferringresistance to a pathogen such as an intracellular pathogen includingviruses and intracellular microorganisms. Cells contemplated as targetsfor the genetic construct of the present invention include animal cells,plant cells, unicellular organisms and microorganisms. Animal cells maybe from primates, hmans, livestock animals, avian speices, fish,reptiles, amphibians and insects and arachnids. Plant cells may be frommonocotyledonas or dicotyledonas plants.

[0071] In one particularly useful embodiment, the peptide, polypeptideor protein encoded by the polynucleotide sequence induces apoptosis orother form of cell death. This is particularly useful as a means offacilitating genetic resistance to viruses, for example, or formediating cell death of particular types of cells.

[0072] In one embodiment, for example, the construct is used tofacilitate resistance to a single stranded DNA (ssDNA) virus. Suchviruses cause considerable damage to the agricultural and horticulturalindustries by infecting important crop and ornamental plants. Two groupsof ssDNA viruses which infect plants are the gemini- and nanoviruses.

[0073] In one embodiment, the flanking sequences recognizable by aviral-derived, replication-facilitating protein or its derivatives orprokaryotic or eukaryotic cell homologues are stem/loop nucleotidestructures. Preferably, the stem/loop structures comprise a shortnucleotide sequence loop of from about 5 to about 20, preferably fromabout 6 to about 15 and most preferably about 9 nucleotides (i.e.nonanucleotide) and which is the site of nicking and ligation by theviral-derived, replication-facilitating protein or its derivatives orprokaryotic or eukaryotic cell homologues. In another embodiment, theflanking sequences are recognized by any protein having cleavage andligation activity. An example of such a protein is topoisomerase. Allthese sequences are referred to herein as “recognition sequences”. Mostpreferably, the recognition sequences are recognized by the “Rep”protein. This protein is derived from members of the geminiviridae andnanoviruses and binds to a 5′ domain on a stem/loop structure comprisingthe recognition sequence. The present invention, however, is not limitedto the use of a stem loop structure although such use is contemplatedherein. The present invention extends to any Rep protein from ageminivirus or nanovirus as well as derivatives thereof or homologuesfrom other viruses or from eukaryotic or prokaryotic cells. Examples ofeukaryotic cells include mammalian, insect, reptilian, amphibian andyeast cells.

[0074] Examples of other recognition sequences or their equivalentsinclude the intergenic regions of BBTV DNA 1-6, the short and longrepeats of TLCV or TYDV. An “intronic sequence” is a sequence ofnucleotides which, following transcription, have the capacity to bespliced out. In certain circumstances, the intronic sequence is notspliced out such as when the presence of the intronic sequence does notadversely affect the functioning of the sequence into which the intronicsequence is inserted.

[0075] The construct of this aspect of the present invention maycomprise the same or substantially the same recognition sequences asflanking sequences, that is, the sequences recognizable by a single Repprotein or its derivatives or homologues or may comprise differentrecognition sequences recognizable by different Rep proteins orderivatives thereof or eukaryotic or prokaryotic cell homologuesthereof. Furthermore, the recognition sequences may be full sequences orpart sequences such as two half intronic sequences.

[0076] The Rep protein may be introduced to a cell such as followingviral infection or be encoded by genetic sequences developmentally ortissue specifically expressed in the animal or plant or organism whichcarries the construct.

[0077] In another preferred embodiment, there is provided a constructcomprising a genetic element flanked by Rep-protein recognitionsequences or functional homologues from other viruses or eukaryotic orprokaryotic cells which facilitate the generation of a circularnucleotide sequence comprising said genetic element in the presence of aRep protein or its functional derivatives or homologues wherein saidRep-protein recognition sequences are adjacent to or inserted within oneor more recognition sequences, said genetic element comprising apolynucleotide sequence operably linked to regulatory sequences requiredto permit expression of said polynucleotide sequence when said geneticelement is contained within a circularized molecule wherein the geneticelement in linear form comprises in the 5′ to 3′ order:—

[0078] a polynucleotide sequence; and

[0079] regulatory sequences to permit expression of said polynucleotidesequence when in circular form,

[0080] such that upon circularization the genetic element comprises theregulatory sequence separated from the polynucleotide sequence by all orpart of a Rep protein-recognition sequence wherein upon expression, saidpolynucleotide sequence encodes an expression product.

[0081] Preferably, the regulatory sequences include or comprise apromoter sequence and optionally a transcription terminator. As statedabove, a recognition sequence includes an extraneous sequence such as anintronic sequence or other splice signal.

[0082] In another embodiment, there is provided a construct comprising agenetic element flanked by a Rep protein-recognition sequences whichfacilitate the generation of a circular nucleotide sequence comprisingsaid genetic element in the presence of a Rep protein or its functionalderivatives or homologues wherein said Rep-protein recognition sequencesare adjacent to or inserted within one or more extraneous sequencesincluding intronic sequences or parts thereof or other splice signals,said genetic element comprising a 3′ portion and a 5′ portion of apromoter separated by a length of a nucleotide sequence to substantiallyprevent functioning of said promoter, said genetic element in linearform comprises in the 5′ to 3′ order:—

[0083] a 3′ portion of said promoter;

[0084] optionally a polynucleotide sequence operably linked to said 3′portion of said promoter; and

[0085] a 5′ portion of said promoter,

[0086] such that upon circularization the genetic element comprises the5′ and 3′ portions of the promoter sequence separated by all or part ofa Rep protein-recognition sequence but which does not inactivate theactivity of the promoter, said circular molecule optionally furthercomprising the promoter operably linked to polynucleotide sequence.

[0087] Alternatively, the promoter is an RNA promoter such as from TMV,TEV or AMV.

[0088] An advantage of such a system is that when the construct is inlinear form and, for example, integrated into a larger nucleotidesequence such as a genome, the promoter sequence is inactive. However,upon circularization, the promoter sequence is reconstituted thuspermitting promoter activity. The optionally present operably linkedpolynucleotide sequence is then expressed.

[0089] Examples of suitable promoters include the cauliflower mosaicvirus 35S promoter. Another useful promoters is the ubiquitin promoter.Generally, monocot promoters such as the ubiquitin promoter require anintronic sequence between the promoter and the start codon of theexpressed exon. Absent this intronic sequence, expression of thepromoter is either very low or completely lacking. The genetic constructof the present invention may be designed such that upon circularization,the intronic sequence comprising the stem loop structure forms anintronic sequence downstream of the ubiquitin promoter thus permittingits operation.

[0090] Other suitable promoters are described below.

[0091] In another preferred embodiment, the present invention provides aconstruct comprising in 5′ to 3′ order first, second, third, fourth,fifth and sixth nucleotide sequences wherein:

[0092] the first and sixth nucleotide sequences may be the same ordifferent and each comprises a Rep protein-recognition sequence capableof being recognized by one or more Rep proteins or derivatives orhomologues thereof such that genetic material flanked by said first andsixth sequences including all or part of said first and sixth sequenceswhen said construct is integrated in a larger nucleotide sequence suchas a genomic sequence, is capable of being excised and circularizedwherein said Rep-protein recognition sequences are adjacent to orinserted within one or more extraneous sequences including intronicsequences or parts thereof or other splice signals;

[0093] the second nucleotide sequence comprises a 3′ portion ofpolynucleotide sequence;

[0094] the third nucleotide sequence is a transcription terminator orfunctional derivative or homologue thereof operably linked to saidsecond sequence;

[0095] the fourth nucleotide sequence is a promoter sequence operablylinked to the fifth nucleotide sequence; and

[0096] the fifth nucleotide sequence is a 5′ portion of a polynucleotidesequence wherein the 5′ and 3′ portions of said polynucleotide sequencerepresent a full coding sequence of said polynucleotide sequence;

[0097] wherein in the presence of one or more Rep proteins, when theconstruct is integrated into a larger nucleotide sequence such as agenomic sequence, a circularized genetic sequence is generated separatefrom said larger nucleotide sequence comprising in order said promotersequence operably linked to a polynucleotide sequence comprising all orpart of the extraneous sequence or other splice signal comprising all orpart of said first and/or sixth nucleotide sequences and a transcriptionterminator sequence.

[0098] In accordance with the above-mentioned aspect of the presentinvention, the first and sixth nucleotide sequences representrecognition sequences for a viral-derived, replication-facilitatingprotein such as Rep or derivatives thereof or eukaroytic or prokaryoticderivatives thereof adjacent to or inserted within an intronic sequenceor other splice signal. The second to fifth nucleotide sequencesrepresent the genetic elements previously defined.

[0099] Yet another aspect of the present invention is directed to agenetic element for use in generating a construct, said genetic elementcomprising in 5′ to 3′ direction, a 3′ portion of a polynucleotidesequence operably linked to a transcription terminator; a promoteroperably linked to a 5′ portion of a polynucleotide sequence whereinupon circularization, the 5′ portion of the polynucleotide sequence isoperably linked to said 3′ portion of the polynucleotide sequenceseparated by all or part of an extraneous sequence or intron sequence orother splice signal.

[0100] The constructs of the present invention have a range ofapplications. In one embodiment, the construct is used to generategenetic resistance in plant cells to ssDNA viruses. The particularviruses for which protection is sought against include but not limitedto geminivirus or nanovirus. In this embodiment, the construct comprisesa “suicide gene”, i.e. a gene encoding a product which induces cellapoptosis, lysis, death or a state of biochemical or physiologicaldormancy. The construct is introduced into a plant cell under conditionsto permit integration into the plant cell genome. A plant is regeneratedfrom the plant cell and propagated when the plant is infected by aparticular ssDNA virus having a Rep protein which recognizes the Repprotein-recognition sequences flanking the genetic element of theconstruct, the construct is excised and recircularizes thusreconstituting the “suicide gene” and facilitating its expression. Thecell then dies or otherwise becomes dormant thus preventing thereplication and release of ssDNA viruses.

[0101] In a particularly preferred embodiment, the present inventionprovides a construct comprising the nucleotide sequence substantially asset forth in SEQ ID NO:31 to SEQ ID NO:36 or a nucleotide sequencehaving 60% similarity to each of SEQ ID NO:31 to SEQ ID NO:36 or anucleotide sequence capable of hybridizing to one or more of SEQ IDNO:31 to SEQ ID NO:36 or a complementary form thereof under lowstringency conditions at 42° C.

[0102] Accordingly, another aspect of the present invention contemplatesa method for generating a transgenic plant or progeny thereof resistantto a ssDNA virus, said method comprising introducing into the genome ofsaid plant a construct comprising in the 5′ to 3′ order, a Repprotein-recognition sequence adjacent to or within an intronic sequenceor other splice signal, a 3′ end portion of a polynucleotide sequence, atranscription terminator or its functional equivalent, a promotersequence operably linked to a 5′ end portion of the polynucleotidesequence wherein the 5′ and 3′ portions of the polynucleotide sequencerepresent the coding region of a peptide, polypeptide or protein capableof inducing cell death or dormancy, and same or different Repprotein-recognition sequences; wherein upon infection of said plantcells by ssDNA virus having a Rep protein which is capable ofrecognizing the flanking Rep protein-recognition sequences, theconstruct is excised and circularizes thus reconstituting saidpolynucleotide sequence in a form which is expressed into a peptide,polypeptide or protein which kills the plant cell or otherwise rendersthe plant cell dormant.

[0103] Another use of the instant construct is to produce male sterileplants. In this embodiment, a gene encoding a Rep protein is placedunder the control of a pollen-specific promoter. A construct comprisingthe above described “suicide gene” is also generated using Repprotein-recognition sequences recognized by the Rep gene under thecontrol of the pollen-specific promoter. When pollen is formed, thepollen-specific promoter is activated thus activating the suicide gene.Pollen cells are then selectively destroyed or rendered dormant.

[0104] Other uses of the construct herein described include introducinggenetic material facilitating a colour change into plants or specifictissue or seeds or other reproductive material of plants. An example ofa genetic sequence facilitating a colour change is a gene encoding anenzyme of a anthocyanin pathway such as a flavonal 3′-hydroxylase,flavonal 3′,5′-hydroxylase, or flavone 3′-synthase.

[0105] In another embodiment, the construct may be flanked by twodifferent Rep protein-recognition sequences, i.e. recognized by twodifferent Rep proteins. One Rep protein may then be encoded by a geneinserted into the plant genome and the other Rep protein may beintroduced by an infecting ssDNA virus. Alternatively, the Rep proteinsmay be encoded by different promoters which are expressed and certaindevelopment stages.

[0106] Although the present invention is particularly described inrelation to plants and ssDNA viruses, the present invention extends tohomologous excision structures and other proteins with site-specificexcision and joining activities from other sources such as non-ssDNAviruses and eukaryotic cells such as insect, mammalian or reptiliancells. Insofar as the present invention relates to plants, the plantsmay be monocotyledonous or dicotyledonous plants.

[0107] The term “plant cell” as used herein includes protoplasts orother cells derived from plants, gamete-producing cells and cells whichregenerate into whole plants. Plant cells include cells in plants aswell as protoplasts or other cells in culture.

[0108] The term “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, cDNA or DNA.

[0109] “Polypeptide”, “peptide” and “protein” are used interchangeablyherein to refer to a polymer of amino acid residues and to variants ofsame.

[0110] Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that the two polynucleotides,sequence comparisons between two (or more) polynucleotides are typicallyperformed by comparing sequences of the two polynucleotides over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment of atleast 6 contiguous positions, usually about 50 to about 100, moreusually about 100 to about 150 in which a sequence is compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. The comparison window may compriseadditions or deletions (i.e., gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. Optimal alignment ofsequences for aligning a comparison window may be conducted bycomputerised implementations of algorithms (GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Drive Madison, Wis., USA) or by inspectionand the best alignment (i.e., resulting in the highest percentagehomology over the comparison window) generated by any of the variousmethods selected. Reference also may be made to the BLAST family ofprograms as for example disclosed by Altschul et al., 1997. A detaileddiscussion of sequence analysis can be found in Unit 19.3 of Ausubel etal., 1994-1998.

[0111] The term “sequence identity” as used herein refers to the extentthat sequences are identical on a nucleotide-by-nucleotide basis or anamino acid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gin, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware.

[0112] Reference herein to a low stringency includes and encompassesfrom at least about 0 to at least about 15% v/v forarnmide and from atleast about 1 M to at least about 2 M salt for hybridization, and atleast about 1 M to at least about 2 M salt for washing conditions.Generally, low stringency is at from about 25-30° C. to about 42° C. Thetemperature may be altered and higher temperatures used to replaceformamide and/or to give alternative stringency conditions. Alternativestringency conditions may be applied where necessary, such as mediumstringency, which includes and encompasses from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization, and at least about 0.5 M to atleast about 0.9 M salt for washing conditions, or high stringency, whichincludes and encompasses from at least about 31% v/v to at least about50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about0.15 M salt for washing conditions. In general, washing is carried outT_(m)=69.3+0.41 (G+C)% (Marmur and Doty, 1962). However, the T_(m) of aduplex DNA decreases by 1° C. with every increase of 1% in the number ofmismatch base pairs (Bonner and Laskey, 1974). Formamide is optional inthese hybridization conditions. Accordingly, particularly preferredlevels of stringency are defined as follows: low stringency is 6×SSCbuffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSCbuffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.;high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of atleast 65° C.

[0113] The term “transformation” means alteration of the genotype of anorganism, for example, a eukaryotic cell, by the introduction of aforeign or endogenous nucleic acid.

[0114] By “vector” is meant a nucleic acid molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, or plantvirus, into which a nucleic acid sequence may be inserted or cloned. Avector preferably contains one or more unique restriction sites and maybe capable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector may be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into a cell,is integrated into the genome of the recipient cell and replicatedtogether with the-chromosome(s) into which it has been integrated. Avector system may comprise a single vector or plasmid, two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon. The choiceof the vector will typically depend on the compatibility of the vectorwith the cell into which the vector is to be introduced. The vector mayalso include a selection marker such as an antibiotic resistance genethat can be used for selection of suitable transformants. Examples ofsuch resistance genes are well known to those of skill in the art.

[0115] The term “gene” is used in its broadest sense and includes cDNAcorresponding to the exons of a gene. Accordingly, reference herein to a“gene” is to be taken to include:—

[0116] (i) a classical genomic gene consisting of transcriptional and/ortranslational regulatory sequences and/or a coding region and/ornon-translated sequences (i.e. introns, 5′- and 3′-untranslatedsequences); or

[0117] (ii) mRNA or cDNA corresponding to the coding regions (i.e.exons) and 5′- and 3′-untranslated sequences of the gene; and/or

[0118] (iii) a structural region corresponding to the coding regions(i.e. exons) optionally further comprising untranslated sequences and/ora heterologous promoter sequence which consists of transcriptionaland/or translational regulatory regions capable of conferring expressioncharacteristics on said structural region.

[0119] The term “gene” is also used to describe synthetic or fusionmolecules encoding all or part of a functional product, in particular, asense or antisense mRNA product or a peptide, oligopeptide orpolypeptide or a biologically-active protein. Reference to a “gene” alsoincludes reference to a “synthetic gene”.

[0120] The term “synthetic gene” refers to a non-naturally occurringgene as hereinbefore defined which preferably comprises at least one ormore transcriptional and/or translational regulatory sequences operablylinked to a structural gene sequence.

[0121] The term “structural gene” shall be taken to refer to anucleotide sequence which is capable of being transmitted to producemRNA and optionally, encodes a peptide, oligopeptide, polypeptide orbiologically active protein molecule. Those skilled in the art will beaware that not all mRNA is capable of being translated into a peptide,oligopeptide, polypeptide or protein, for example, if the mRNA lacks afunctional translation start signal or alternatively, if the mRNA isantisense mRNA. The present invention clearly encompasses syntheticgenes comprising nucleotide sequences which are not capable of encodingpeptides, oligopeptides, polypeptides or biologically-active proteins.In particular, the present inventors have found that such syntheticgenes may be advantageous in modifying target gene expression in cells,tissues or organs of a eukaryotic organism.

[0122] The term “structural gene region” refers to that part of asynthetic gene which is expressed in a cell, tissue or organ under thecontrol of a promoter sequence to which it is operably connected. Astructural gene region may be operably under the control of a singlepromoter sequence or multiple promoter sequences. Accordingly, thestructural gene region of a synthetic gene may comprise a nucleotidesequence which is capable of encoding an amino acid sequence or iscomplementary thereto. In this regard, a structural gene region which isused in the performance of the instant invention may also comprise anucleotide sequence which encodes an amino acid sequence yet lacks afunctional translation initiation codon and/or a functional translationstop codon and, as a consequence, does not comprise a complete openreading frame. In the present context, the term “structural gene region”also extends to a non-coding nucleotide sequences, such as 5′-upstreamor 3′-downstream sequences of a gene which would not normally betranslated in a eukaryotic cell which expresses said gene.

[0123] Accordingly, in the context of the present invention, astructural gene region may also comprise a fusion between two or moreopen reading frames of the same or different genes. In such embodiments,the invention may be used to modulate the expression of one gene, bytargeting different non-contiguous regions thereof or alternatively, tosimultaneously modulate the expression of several different genes,including different genes of a multigene family. In the case of a fusionnucleic acid molecule which is non-endogenous to a eukaryotic cell andin particular comprises two or more nucleotide sequences derived from aviral pathogen, the fusion may provide the added advantage of conferringsimultaneous immunity or protection against several pathogens, bytargeting the expression of genes in said several pathogens.Alternatively or in addition, the fusion may provide more effectiveimmunity against any pathogen by targeting the expression of more thanone gene of that pathogen.

[0124] Particularly preferred structural gene regions according to thisaspect of the invention are those which include at least onetranslatable open reading frame, more preferably further including atranslational start codon located at the 5′-end of said open readingframe, albeit not necessarily at the 5′-terminus of said structural generegion. In this regard, notwithstanding that the structural gene regionmay comprise at least one translatable open reading frame and/or AUG orATG translational start codon, the including of such sequences in no waysuggest that the present invention requires translation of theintroduced nucleic acid molecule to occur in order to modulate theexpression of the target gene. Whilst not being bound by any theory ormode of action, the inclusion of at least one translatable open readingframe and/or translational start codon in the subject nucleic acidmolecule may serve to increase stability of the mRNA transcriptionproduct thereof, thereby improving the efficiency of the invention.

[0125] The optimum number of structural gene sequences which may beinvolved in the synthetic gene of the present invention will varyconsiderably, depending upon the length of each of said structural genesequences, their orientation and degree of identity to each other. Forexample, those skilled in the art will be aware of the inherentinstability of palindromic nucleotide sequences in vivo and thedifficulties associated with constructing long synthetic genescomprising inverted repeated nucleotide sequences because of thetendency for such sequences to recombine in vivo. Notwithstanding suchdifficulties, the optimum number of structural gene sequences to beincluded in the synthetic genes of the present invention may bedetermined empirically by those skilled in the art, without any undueexperimentation and by following standard procedures such as theconstruction of the synthetic gene of the invention usingrecombinase-deficient cell lines, reducing the number of repeatedsequences to a level which eliminates or minimizes recombination eventsand by keeping the total length of the multiple structural gene sequenceto an acceptable limit, preferably no more than 5-10 kb, more preferablyno more than 2-5 kb and even more preferably no more than 0.5-2.0 kb inlength.

[0126] For expression in eukaryotic cells, the construct generallycomprises, in addition to the polynucleotide sequence, a promoter andoptionally other regulatory sequences designed to facilitate expressionof the polynucleotide sequence.

[0127] Reference herein to a “promoter” is to be taken in its broadestcontext and includes the transcriptional regulatory sequences of aclassical genomic gene, including the TATA box which is required foraccurate transcription initiation, with or without a CCAAT box sequenceand additional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner. Apromoter is usually, but not necessarily, positioned upstream or 5′, ora structural gene region, the expression of which it regulates.Furthermore, the regulatory elements comprising a promoter are usuallypositioned within 2 kb of the start site of transcription of the gene.

[0128] In the present context, the term “promoter” is also used todescribe a synthetic or fusion molecule, or derivative which confers,activates or enhances expression of a nucleic acid molecule in a cell.

[0129] Preferred promoters may contain additional copies of one or morespecific regulatory elements, to further enhance expression of the sensemolecule and/or to alter the spatial expression and/or temporalexpression of said sense molecule. For example, regulatory elementswhich confer copper inducibility may be placed adjacent to aheterologous promoter sequence driving expression of a sense molecule,thereby conferring copper inducibility on the expression of saidmolecules.

[0130] Placing a nucleic acid molecule under the regulatory control of apromoter sequence means positioning the said molecule such thatexpression is controlled by the promoter sequence. Promoters aregenerally positioned 5′ (upstream) to the genes that they control. Inthe construction of heterologous promoter/structural gene combinations,it is generally preferred to position the promoter at a distance fromthe gene transcription start site that is approximately the same as thedistance between that promoter and the gene it controls in its naturalsetting, i.e. the gene from which the promoter is derived. As is knownin the art, some variation in this distance can be accommodated withoutloss of promoter function. Similarly, the preferred positioning of aregulatory sequence element with respect to a heterologous gene to beplaced under its control is defined by the positioning of the element inits natural setting, i.e. the genes from which it is derived. Again, asis known in the art, some variation in this distance can also occur.

[0131] Examples of promoters suitable for use in the synthetic genes ofthe present invention include viral, fungal, bacterial, animal and plantderived promoters capable of functioning in plant, animal, insect,fungal, yeast or bacterial cells. The promoter may regulate theexpression of the structural gene component constitutively, ordifferentially with respect to cell, the tissue or organ in whichexpression occurs or, with respect to the developmental stage at whichexpression occurs, or in response to external stimuli such asphysiological stresses, or pathogens, or metal ions, amongst others.

[0132] Preferably, the promoter is capable of regulating expression of anucleic acid molecule in a eukaryotic cell, tissue or organ, at leastduring the period of time over which the target gene is expressedtherein and more preferably also immediately preceding the commencementof detectable expression of the target gene in said cell, tissue ororgan.

[0133] Accordingly, strong constitutive promoters are particularlyuseful for the purposes of the present invention or promoters which maybe induced by virus infection or the commencement of target geneexpression.

[0134] Plant-operable and animal-operable promoters are particularlypreferred for use in the construct of the present invention. Examples ofpreferred promoters include the viral promoters such as bacteriophage T7promoter, bacteriophage T3 promoter, SP6 promoter, bacterial promoterssuch as lac operator-promoter, tac promoter, viral promotors such asSV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IEpromoter or plant viral promoters such as CaMV 35S promoter, SCSVpromoter, SCBV promoter and the like.

[0135] In consideration of the preferred requirement for high-levelexpression which coincides with expression of the target gene orprecedes expression of the target gene, it is highly desirable that thepromoter sequence is a constitutive strong promoter in the host ofinterest such as the CMV-IE promoter or the SV40 early promotersequence, the SV40 late promoter sequence for mammalian cells and, theCaMV 35S promoter, or the SCBV promoter in certain plant cells, amongstothers. Those skilled in the art will readily be aware of additionalpromoter sequences other than those specifically described.

[0136] In the present context, the terms “in operable connection with”or “operably under the control” or similar shall be taken to indicatethat expression of the structural gene region or multiple structuralgene region is under the control of the promoter sequence with which itis spatially connected; in a cell, tissue, organ or whole organism.

[0137] The construct preferably contains additional regulatory elementsfor efficient transcription, for example, a transcription terminationsequence.

[0138] The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences generally containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active inplant cells are known and described in the literature. They may beisolated from bacteria, fungi, viruses, animals and/or plants orsynthesized de novo.

[0139] As with promoter sequences, the terminator may be any terminatorsequence which is operable in the cells, tissues or organs in which itis intended to be used.

[0140] Examples of terminators particularly suitable for use in thesynthetic genes of the present invention include the SV40polyadenylation signal, the HSV TK polyadenylation signal, the CYC1terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) geneterminator of Agrobacterium tumefaciens, the terminator of thecauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator fromZea mays, the Rubisco small subunit gene (SSU) gene terminatorsequences, subclover stunt virus (SCSV) gene sequence terminators, anyrho-independent E. coli terminator, or the lacZ alpha terminator,amongst others.

[0141] In a particularly preferred embodiment, the terminator is theSV40 polyadenylation signal or the HSV TK polyadenylation signal whichare operable in animal cells, tissues and organs, octopine synthase(OCS) or nopaline synthase (NOS) terminator active in plant cells,tissue or organs, or the lacZ alpha terminator which is active inprokaryotic cells.

[0142] Those skilled in the art will be aware of additional terminatorsequences which may be suitable for use in performing the invention.Such sequences may readily be used without any undue experimentation.

[0143] Means for introducing (i.e. transfecting or transforming) cellswith the constructs are well-known to those skilled in the art.

[0144] The constructs described supra are capable of being modifiedfurther, for example, by the inclusion of marker nucleotide sequencesencoding a detectable marker enzyme or a functional analogue orderivative thereof, to facilitate detection of the synthetic gene in acell, tissue or organ in which it is expressed. According to thisembodiment, the marker nucleotide sequences will be present in atranslatable format and expressed, for example, as a fusion polypeptidewith the translation product(s) of any one or more of the structuralgenes or alternatively as a non-fusion polypeptide.

[0145] Those skilled in the art will be aware of how to produce thesynthetic genes described herein and of the requirements for obtainingthe expression thereof, when so desired, in a specific cell or cell-typeunder the conditions desired. In particular, it will be known to thoseskilled in the art that the genetic manipulations required to performthe present invention may require the propagation of a genetic constructdescribed herein or a derivative thereof in a prokaryotic cell such asan E. coli cell or a plant cell or an animal cell.

[0146] The constructs of the present invention may be introduced to asuitable cell, tissue or organ without modification as linear DNA,optionally contained within a suitable carrier, such as a cell, virusparticle or liposome, amongst others. To produce a genetic construct,the synthetic gene of the invention is inserted into a suitable vectoror opisome molecule, such as a bacteriophage vector, viral vector or aplasmid, cosmid or artificial chromosome vector which is capable ofbeing maintained and/or replicated and/or expressed in the host cell,tissue or organ into which it is subsequently introduced.

[0147] Accordingly, a further aspect of the invention provides a geneticconstruct which at least comprises a genetic element as herein describedand one or more origins of replication and/or selectable marker genesequences.

[0148] Genetic constructs are particularly suitable for thetransformation of a eukaryotic cell to introduce novel genetic traitsthereto, in addition to the provision of resistance characteristics toviral pathogens. Such additional novel traits may be introduced in aseparate genetic construct or, alternatively, on the same geneticconstruct which comprises the synthetic genes described herein. Thoseskilled in the art will recognize the significant advantages, inparticular in terms of reduced genetic manipulations and tissue culturerequirements and increased cost-effectiveness, of including geneticsequences which encode such additional traits and the synthetic genesdescribed herein in a single genetic construct.

[0149] Usually, an origin of replication or a selectable marker genesuitable for use in bacteria is physically-separated from those geneticsequences contained in the genetic construct which are intended to beexpressed or transferred to a eukaryotic cell, or integrated into thegenome of a eukaryotic cell.

[0150] As used herein, the term “selectable marker gene” includes anygene which confers a phenotype on a cell on which it is expressed tofacilitate the identification and/or selection of cells which aretransfected or transformed with a genetic construct of the invention ora derivative thereof.

[0151] Suitable selectable marker genes contemplated herein include theampicillin-resistance gene (Amp^(r)), tetracycline-resistance gene(Tc^(r)), bacterial kanamycin-resistance gene (Kan^(r)), is the zeocinresistance gene (Zeocin is a drug of the bleomycin family which is trademark of In Vitrogen Corporation), the A URI-C gene which confersresistance to the antibiotic aureobasidin A, phosphinothricin-resistancegene, neomycin phosphotransferase gen (nptII), hygromycin-resistancegene, β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase(CAT) gene, green fluorescent protein-encoding gene or the luciferasegene, amongst others.

[0152] Preferably, the selectable marker gene is the nptII gene orKan^(r) gene or green fluorescent protein (GFP)-encoding gene.

[0153] Those skilled in the art will be aware of other selectable markergenes useful in the performance of the present invention and the subjectinvention is not limited by the nature of the selectable marker gene.

[0154] The present invention extends to all genetic constructsessentially as described herein, which include further genetic sequencesintended for the maintenance and/or replication of said geneticconstruct in prokaryotes or eukaryotes and/or the integration of saidgenetic construct or a part thereof into the genome of a eukaryotic cellor organism.

[0155] Standard methods described supra may be used to introduce theconstructs into the cell, tissue or organ, for example,liposome-mediated transfection or transformation, transformation ofcells with attenuated virus particles or bacterial cells, cell mating,transformation or transfection procedures known to those skilled in theart.

[0156] Additional means for introducing recombinant DNA into planttissue or cells include, but are not limited to, transformation usingCaCl₂ and variations thereof, direct DNA uptake into protoplasts,PEG-mediated uptake to protoplasts, microparticle bombardment,electroporation, microinjection of DNA, microparticle bombardment oftissue explant or cells, vacuum-infiltration of tissue with nucleicacid, or in the case of plants, T-DNA-mediate transfer fromAgrobacterium to the plant tissue.

[0157] For microparticle bombardment of cells, a microparticle ispropelled into a cell to produce a transformed cell. Any suitableballistic cell transformation methodology and apparatus can be used inperforming the present invention. Exemplary apparatus and procedures aredisclosed by Stomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf(U.S. Pat. No. 4,945,050). When using ballistic transformationprocedures, the genetic construct may incorporate a plasmid capable ofreplicating in the cell to be transformed.

[0158] Examples of microparticles suitable for use in such systemsinclude 1 to 5 μm gold spheres. The DNA construct may be deposited onthe microparticle by any suitable technique, such as by precipitation.

[0159] In a further embodiment of the present invention, the geneticconstructs described herein are adapted for integration into the genomeof a cell in which it is expressed. Those skilled in the art will beaware that, in order to achieve integration of a genetic sequence orgenetic construct into the genome of a host cell, certain additionalgenetic sequences may be required. In the case of plants, left and rightborder sequences from the T-DNA of the Agrobacterium tumefaciens Tiplasmid will generally be required.

[0160] The present invention further extends to an isolated cell, tissueor organ comprising the constructs or parts thereof The presentinvention extends further to regenerated tissues, organs and wholeorganisms derived from said cells, tissues and organs and to propagulesand progeny thereof as well as seeds and other reproductive material.

[0161] For example, plants may be regenerated from transformed plantcells or tissues or organs on hormone-containing media and theregenerated plants may take a variety of forms, such as chimeras oftransformed cells and non-transformed cells; clonal transformants (e.g.all cells transformed to contain the expression cassette); grafts oftransformed and untransformed tissue (e.g. a transformed root stockgrafted to an untransformed scion in citrus species). Transformed plantsmay be propagated by a variety of means, such as by clonal propagationor classical breeding techniques. For example, a first generation (orT1) transformed plants may be selfed to give homozygous secondgeneration (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques.

[0162] In another embodiment, the construct is used to induce modulationof expression of a target genetic sequence. For example, a construct,when in linear form, comprises in the 5′ to 3′ direction an antisensesequence, a promoter and a sense sequence. These elements are thenflanked by viral-derived, replication-faciliating protein recognitionsequences (e.g. stem loop) or mammalian or microbial homologues. Aterminator sequence is located outside the recognition-sequence flankedregion. Upon replicative release, a polynucleotide sequence comprisingantisense and sense forms of a target genetic sequence is produced.

[0163] The resulting polynucleotide sequence may then form a hair-pinloop. The construct may be varied to produce tandem or multiple repeats,inverts or combinations thereof Such constructs are useful for genesilencing in plant and animal cells. The order of elements in the linearform is not critical. For example, the location of the sense andantisense sequences may be exchanged. An example of one suitableconstruct is shown in FIG. 27.

[0164] The present invention is further described by the followingnon-limiting Examples.

EXAMPLE 1

[0165] Expression Vectors Based on BBTV

[0166] A series of expression vectors were constructed which contain thecauliflower mosaic virus 35S promoter (35S) driving expression of thegene encoding barnase, into which the intron from the potatolight-inducible tissue-specific ST-LS1 gene was introduced(NT-INTRON-CT). The terminator was derived from either the gene encodingnopaline synthase (nos) or the major open-reading frame (ORF) of BBTVDNA-6 (BT6).

[0167] The constructs were assembled using PCR with overlapping primers(Table 1) and cloned into pGEM-T vectors using standard techniques knownin the art. The expression vector pTBN was constructed and is shown inFIG. 1. pTBN (SEQ ID NO:66) represents the backbone upon which otherexpression vectors were constructed. Primer names and binding sites areindicated, as is the size of each sequence. pTBN (like the otherconstructs) was amplified in a step-wise manner. Initially, all threefragments were separately amplified (i.e. 35S, NT-INTRON-CT and nos).The entire sequence was then amplified by mixing each of the fragmentsin a PCR with primers +1 and −4.

[0168] pTBN6 (SEQ ID NO:67) shown in FIG. 2 contains a 624 bp regioncontaining the CR-SL and CR-M of BBTV DNA-6 (6I), inserted into theintron.

[0169] pTBN1 (SEQ ID NO:68) shown in FIG. 3 contains 163 bp regioncontaining the CR-SL and CR-M of BBTV DNA-1 (11), inserted into theintron, identically to pTBN6. The nos terminator was replaced by the 126bp terminator from BBTV DNA-6 large ORF.

[0170] Recircularization vectors are based upon pTBN6 and pTBN1. Theyare flanked by Rep recognition sequences and designed to recircularizeand subseqently be transcriptionally active, only in the presence of theBBTV Rep. Three different vectors were made: pRTBN6, pRTBN1 andpRTBN1/6.

[0171] pRTBN6 shown in FIG. 4 was constructed from pTBN6. Two fragmentswere amplified using +5 and −4 and +1 and 6 respectively. These werecloned into pGEM-T and sub-cloned to create pRTBN6.

[0172] pRTBN1 shown in FIG. 5 was constructed from pTBN1 in a similarfashion to pRTBN6.

[0173] pRTBN1/6 shown in FIG. 6 was a hybrid of pRTBN1 and pRTBN6.

[0174] Untranslatable vectors were also constructed for each of theconstructs mentioned above (except pTBN). In these vectors, the startcodon of the barnase gene was deleted using the +11 primer (refer toTable 1). The constructs were named pUBN6, pUBN1, pRUBN6 and pRUBN1.TABLE 1 Oligonucleotide primer sequences Primer Name Sequence (5′-3′) +13TS1-H AAGCTTCATGGAGTCAAAGA (SEQ ID NO:1) +2 35S1-BTCATTTGGAGAGGATCCATGGCACAGGTT (SEQ ID NO:2) −2 B2-35SAACCTGTGCCATGGATCCTCTCCAAATGA (SEQ ID NO:3) +3 B1-NAAAATCAGATAAGAGCTCGATCGTTCAAA (SEQ ID NO:4) −3 N2-BTTTGAACGATCGAGCTCTTATCTGATTTT (SEQ ID NO:5) −4 NOS4-HAAGCTTTTCGCCATTCAGGCTGC (SEQ ID NO:6) +5 B3-6TATCATTAATTAGTAAGTTGTGCTGTAA (SEQ ID NO:7) −5 6-B4TTACAGCACAACTTACTAATTAATGATA (SEQ ID NO:8) +6 6-B3GGAAGGCAGAAGCGAGTAATATAATATT (SEQ ID NO:9) −6 B4-6AATATTATATTACTCGCTTCTGCCTTCC (SEQ ID NO:10) +7 B5-IATCATTAATTAGTCACACTATGACAAAAG (SEQ ID NO:11) −7 I-B6TTGTCATAGTGTGACTAATTAATGATAAT (SEQ ID NO:12) +8 I-B5GACATTTGCATCAGTAATATAATATTTCA (SEQ ID NO:13) −8 B6-IAATATTATATTACTGATGCAAATGTCCCG (SEQ ID NO:14) +9 B7-6TTCAGATAAGAGCTCAGTAACAGCAACAAC (SEQ ID NO:15) −9 6T2-BGCTGTTACTGAGCTCTTATCTGATCTTTG (SEQ ID NO:16) −10 6T4-HAAGCTTATTTCCCAAATATACGT (SEQ ID NO:17) +11 UNTBarn GGATCCGCACAGGTTATCAAC(SEQ ID NO:18)

EXAMPLE 2

[0175] Expression Vectors Based on TYDV

[0176] (a) Construction of an Intron-Containing GUS Reporter GeneExpression Cassette

[0177] The vector pCAMBIA 2301 was obtained from CAMBIA (Canberra,Australia). This vector contains a 189 bp catalase intron within the 5′portion of the uidA coding region. The CAMV 35S promoter region (800bp), uidA coding region, and nos terminator were removed from pCAMBIA2301 as a HindIII/SphI fragment and inserted into similarly digestedpGEM-T (Promega) vector. The subsequent construct was designatedpGEM-2301. The 800 bp CaMV 35S promoter was replaced with the stronger530 bp CaMV 35S promoter by NotI/BglII digestion and ligation. Thesubsequent vector was designated p35S-2301 (FIG. 7) and served as thetemplate for all subsequent cloning steps.

[0178] (b) Isolation of the Tobacco Yellow Dwarf Mastrevirus (TYDV)Large Intergenic Region and Insertion into the Catalase Intron ofp35S-2301

[0179] A 272 bp fragment incorporating the large intergenic region (LIR)(nt +1 to nt +272) of TYDV (Genbank Acc M81103) was amplified fromTYDV-infected tobacco leaf tissue by PCR using primers LIR-F and LIR-R(see FIG. 1). This fragment was designated LIR.

[0180] Primers: LIR-F 5′-GCTCTTCCTGCAG GCGGCCGCATTAAGGCTCAAGTACCGTA3′[SEQ ID NO:19] LIR-R 5′-GCTCTTCGTCGAC GAATTCATTTTCAACTTTGGGATGTCAC-3′[SEQ ID NO:20]

[0181] The segment comprising the CaMV 35S promoter (530 bp), uidA1^(st) exon (19 bp), and 5′ half of the catalase intron (83 bp) wasamplified from p3⁵S-²301 plasmid DNA by PCR, using primers 35S-IE andCAT-A. This fragment was designated CAT-A.

[0182] Primers: 35S-IE 5′-GAATT CCATGGAGTCAAAGATTCA-3′ [SEQ ID NO:21]CAT-A 5′-GCCCGCTGCAGAGTTTAAAGAAAGATCAAAGC-3′ [SEQ ID NO:22]

[0183] The segment comprising the 3′ half of the catalase intron (106bp) and uidA 2^(nd) exon (1809 bp) was amplified from p35S-2301 plasmidDNA by PCR, using primers CAT-B and GUS-BstEII. This fragment wasdesignated CAT-B.

[0184] Primers: CAT-B 5′-GCCCCGGTCGACGATCTATTTTTTAATTGATTGG-3′ [SEQ IDNO:23] GUS-BstEII 5′-TTCGAGCTGGTCACCTGTAATTCACACGTGGTG-3′ [SEQ ID NO:24]

[0185] The resulting PCR products were cloned into pGEM-T and thenucleotide sequence verified. Ultimately, each fragment was excised(CAT-A using EcoRI/PstI, LIR using PstI/SalI, and CAT-B usingSalI/BstEII) from pGEM-T, and ligated together into EcoRI/BstElIdigested p35S-2301 to create the plasmid pTEST1 (FIG. 8).

[0186] (c) To Determine Whether GUS Expression is Affected by Insertionof the TYDV LIR into the Catalase Intron

[0187] In order to determine whether GUS expression, and thereforeintron splicing, was affected by insertion of the TYDV LIR into thecatalase intron of p35S-2301, constructs were bombarded into embryogenicbanana cells and GUS activity transiently assayed. Test plasmid pTEST1and positive control plasmid p35S-2301 were coated onto 1 μm goldparticles and biolistically introduced into 5 day old banana (Musa spp.cv. “Ladyfinger” AAA) embryogenic cells according to Becker et al.(2000). Two days post-bombardment, cells were harvested and GUS activityassayed histochemically (Jefferson et al., 1987).

[0188] No endogenous GUS activity was observed in non-bombarded cells.Strong GUS activity, evident as bright blue staining cell foci, wasobserved from cells bombarded with the positive control plasmidp35S-2301. In contrast, GUS expression from cells bombarded with pTESTl,was lower (about 5-fold) as determined by number and intensity of bluestaining cell foci. This result suggested that insertion of the TYDV LIRinto the catalase intron of p35S-2301 does not abolish GUS expression,but may affect intron processing to some degree.

[0189] (d) Identification of Cryptic Intron Splice Sites within the TYDVLIR

[0190] In order to determine whether the TYDV LIR contained potentialcryptic intron splice sites, which may affect pre-mRNA processing, cDNAwas synthesized from RNA extracts derived from cells bombarded withp35S-2301 and pTEST1. Total RNA was isolated from banana cells two dayspost bombardment with p35S-2301 and pTESTl using the method of Chang etal. (1993) Complementary DNA was synthesized from total RNA using theprimer uidA2. This cDNA served as a template for a nested PCR usingprimers uidA1 and uidA3. The resulting PCR products were cloned intopGEM-T and sequenced. Sequencing identified two potential sites withinthe TYDV LIR, which may contribute to aberrant splicing of the catalaseintron from the uidA coding region pre-mRNA. The first sequence,CTGCAG∇GC, located within the primer LIR-F used to isolated the TYDVLIR, bears strong similarity to the consensus 3′ splice site(T(10×)GCAG∇GT). The second sequence, TA∇GTGAGT (nt +43 to nt +50),shares some similarity to the consensus 5′ splice site (AG∇GTAAGT).

[0191] Primers: uidA1 5′-CCATGGTAGATCTGAGGG-3′ [SEQ ID NO:25] uidA25′-TACGTACACTTTTCCCGGCAATAAC- [SEQ ID NO:26] 3′ uidA35′-GTAACGCGCTTTCCCACCAACGC-3′ [SEQ ID NO:27]

[0192] (e) Removal of the 3′ Cryptic Intron Splice Site from the TYDVLIR

[0193] Of the two cryptic intron splice sites identified, the first(CTGCAGGC) was considered the most significant due to its location inrelation to the 5′ catalase intron splice site. In order to remove thissequence from the TYDV LIR, a new primer, LIR-Xho, was designedincorporating a XhoI site in place of the original PstI and NotIrestriction sites.

[0194] The TYDV LIR was re-amplified from pTEST1 plasmid DNA by PCRusing primers LIR-Xho and LIR-R. This fragment was designated LIR-X.Similarly, the fragment comprising the CaMV 35S promoter (530 bp), uidA1^(st) exon (19 bp) and 5′ half of the catalase intron (83 bp) wasre-amplified from pTEST1 plasmid DNA by PCR using primers FUP andCAT-Xho. This fragment was designated CAT-X. Both PCR products werecloned into pGEM-T and their sequences verified. Ultimately, PCRfragments were excised from pGEM-T (LIR-X using XhoI/SalI and CAT-Xusing PstI/XhoI) and ligated into PstI/SalI digested pTEST1, to replacethe original inserts. This construct was designated pTEST2 (FIG. 9).Removal of the cryptic intron splice site in pTEST2 generated higherlevels of GUS expression than pTEST1 due to a reduction in aberrantsplicing and improved mRNA processing.

[0195] Primers: LIR-Xho 5′-CTCGAGATTAAGGCTCAAGTACCG [SEQ ID NO:28] TA-3′CAT-Xho 5′-AGTTTAAAGAAAGATCAAAGC-3′ [SEQ ID NO:29] FUP5′-AATTAACCCTCACTAAAGGG-3′ [SEQ ID NO:30]

[0196] (f) Construction of the Rep-Activatable GUS Expression Vector

[0197] A 229 bp fragment incorporating the TYDV small intergenic region(SIR) (nt +1275 to nt +1504) was amplifed from TYDV-infected tobaccoleaf tissue by PCR using primers SIR-F and SIR-R (see FIG. 2). Theresulting PCR product was cloned into pGEM-T and the nucleotide sequenceverified. This plasmid was designated pGEM-SIR. The TYDV SIR was excisedfrom pGEM-SIR as a SphI fragment and inserted into the unique SphI site,downstream of the nos terminator, in pTEST1. This construct wasdesignated pTEST1-SIR.

[0198] Primers: SIR-F 5′-GCATGCAAGAGTTGGCGGTAGATTCC [SEQ ID NO:31]GCATGT-3′ SIR-R 5′-GCTCTTCGCGGCCGCGCTCCTGAATCGTCGAGTCA-3′ [SEQ ID NO:32]

[0199] The CaMV 35S promoter, uidA 1^(st) exon, catalase intron 5′ half,and TYDV LIR were excised from pTEST2 as a NotI/SalI fragment andinserted into a similarly-digested pGEM-T vector. The subsequent clonewas designated pGEM-CATX. The TYDV LIR, catalase intron 3′ half, uidA2^(nd) exon, nos terminator, and TYDV SIR were excised from pTEST1-SIRas a NotI fragment and inserted into the unique NotI site in pGEM-CATX.This construct was designated pTEST3 (FIG. 10). The activatable GUSexpression cassette was subsequently excised from pTEST3 by SacI/ApaIdigestion, and inserted into the SacI/ApaI restriction sites locatedupstream of the nos pro-NPTII-nos ter cassette in the binary plasmidpART27 (Gleave 1992). This construct was designated pTEST4 (FIG. 11).The vector pTEST4 was introduced into Agrobacterium tumefaciens(LBA4404) by electroporation using the method of Singh et al. (1993).

[0200] (g) Construction of the Infectious TYDV 1.1 mer

[0201] Two overlapping fragments of the TYDV genome were amplified fromTYDV-infected tobacco leaf tissue by PCR using primer pairs LIR-F/SIR-R(SEQ ID NO:19/SEQ ID NO:32) and LIR-R/TYD-3F (SEQ ID NO:20/SEQ IDNO:33). The resulting PCR products (TYD-R and TYD-L, respectively) werecloned into pGEM-T and their sequences verified. These plasmids weredesignated pGEM-TYD-R and pGEM-TYD-L, respectively. A 1659 bp fragmentof the TYDV genome was excised from pGEM-TYD-L by digestion with EcoRI,and inserted into the unique EcoRI site in pGEM-TYD-R. This constructwas designated pGEM-TYDV1.1 mer. The TYDV 1.1 mer was excised frompGEM-TYDV1.1 mer by SalI/EcoRI partial digestion and inserted intosimilarly digested pBI101.3 vector (Clontech), to replace the uidA geneand nos terminator. This construct was designated pBI-TYDV1.1 mer (FIG.12). The vector pBI-TYDV1.1 mer was introduced into Agrobacteriumtumefaciens (LBA4404) by electroporation using the method of Singh etal. (1993).

[0202] Primers: TYD-3F 5′-TTTAAACGTTTAGGGGTTAGCA-3′ [SEQ ID NO:33]

[0203] (h) Construction of the CaMV 35S-TYDV Rep Fusion

[0204] The complete Rep (including RepA) gene of TYDV (nt +2580 to nt+1481) was amplified from TYDV-infected tobacco leaf tissue by PCR usingprimers TYDVRepF and TYDVRepR. The resulting PCR product was directlycloned into the SmaI site located between the CaMV 35S promoter (530 bp)and CaMV 35S terminator (200 bp) in pDH51 (Pietrzak et al., 1986). Thisconstruct was designated p35S-Rep (FIG. 13).

[0205] Primers: TYDVRepF 5′-TCAGTGACTCGACGATTC-3′ [SEQ ID NO:34]TYDVRepR 5′-TTAATATGCCTTCAGCCC-3′ [SEQ ID NO:35]

EXAMPLE 3

[0206] GUS Expression Assays using TYDV Vectors

[0207] (a) Transient Rep-Activated Expression of GUS in Dicot andMonocot Cells

[0208] Tobacco (NT-1) cells are maintained essentially as described byAn (1985), and prepared for microparticle bombardment as detailed byDugdale et al. (1998). Banana (Musa spp. Cv. “Ladyfinger” AAA)embryogenic cell suspensions were prepared as previously described.Coating of gold particles and biolistic parameters were essentially asdescribed by Dugdale et al. (1998) or Becker et al. (2000).

[0209] Plasmids used for this study included:—

[0210] (i) p35S-2301 as positive control (FIG. 7),

[0211] (ii) pTEST3 (FIG. 10),

[0212] (iii) p35S-Rep (FIG. 13), and

[0213] (iv) pTEST3 and p35S-Rep.

[0214] Five plates of both cell lines are bombarded for each of the fourplasmid combinations. Cells are harvested three days post-bombardmentand GUS activity assayed histochemically and/or fluorometrically(Jefferson et al., 1987).

[0215] No endogenous GUS activity is observed in non-bombarded cells.Strong GUS activity, evident as bright blue staining cell foci, isobserved from cells bombarded with the positive control plasmidp35S-2301. No GUS expression is observed from cells bombarded witheither p35S-Rep or pTEST3. In contrast, cells bombarded with bothp35S-Rep and pTEST3 stain intensely blue, greater than that obtainedwith the positive control plasmid p35S-2301. This result suggests thatonly upon addition of the TYDV Rep in trans does the GUS expressioncassette in pTEST3 become activated.

[0216] (b) Detection of Rep-Assisted Nicking, Joining and Replication ofthe GUS Multicopy Plant Episome (MPE)

[0217] Detection of the TYDV-based GUS MPEs is achieved using a PCRapproach. Primers uidA1 (SEQ ID NO:25) and uidA3 (SEQ ID NO:27) amplifya fragment of the uidA gene spanning the catalase intron. Only uponRep-assisted release of the TYDV-based MPE from the plasmid pTEST3 doesthis primer combination generate a 600 bp product (including 140 bp ofthe uidA gene, 190 bp of the catalase intron and 270 bp of the TYDV LIR)in a PCR.

[0218] Tobacco NT-1 and banana cells are bombarded with each of the fourplasmid combinations listed above. Three days post-bombardment, cellsare harvested and total gDNA extracted using the method of Stewart andVia (1993). Total gDNA (1 μg) is used as a template for a PCR withprimers uidA1 (SEQ ID NO:25) and uidA3 (SEQ ID NO:27). PCR products areelectrophoresed through a 1.5% w/v agarose gel. A 330 bp product isobtained from gDNA of cells bombarded with the control plasmid p35S-2301(FIG. 7). This product corresponded to the uidA and catalase intronsequence from the input plasmid DNA. No PCR product is obtained fromgDNA of non-bombarded cells, or cells bombarded with pTEST3 or p35S-Repalone. A 600 bp product is obtained from gDNA of cells bombarded withboth pTEST3 and p35S-Rep. This result supports previous GUShistochemical assays and suggests the TYDV-based MPEs are only generatedin cells bombarded with both pTEST3 and p35S-Rep.

[0219] Replication of the TYDV-based MPEs is assessed by Southernhybridization. Using this approach, multimeric forms of the MPE,indicative of rolling circle replication, are detected by hybridisation.Total gDNA (20 ug) from cells bombarded with each of the four plasmidcombinations, are electrophoresed through a 1.5% w/v agarose gel. DNA istransferred to a nylon membrane (Roche) by the method of Southern(1975). A 600 bp DIG-labelled probe, specific for the uidA and catalaseintron, is amplified by PCR using primers uidA1 (SEQ ID NO:25) and uidA3(SEQ ID NO:27). The uidA-specific probe is hybridised with the nylonmembrane at 42° C. in DIG Easy-Hyb solution (Roche) and signal detectedusing CDP-Star substrate (Roche) according to manufacturer'sinstructions. Characteristic supercoiled, linear, and open circularforms and higher molecular weight multimeric forms of the TYVD-basedMPEs are only detected in gDNA from plant cells bombarded with bothpTEST3 and p35S-2301. Together these results confirm that, when providedin trans, the TYDV Rep is capable of nicking, joining, and replicatingthe TYDV-based MPE in both monocotyledonous and dicotyledonous celltypes. Further, these results suggest that uidA expression from theplasmid pTEST3 is only activated upon addition of the TYDV Rep, and theaddition results in significantly higher expression than anon-replicating GUS expression cassette (p35S-2301).

EXAMPLE 4

[0220] Stable Transformation of a Monocotyledonous and a DicotyledonousPlant with the Rep-Activatable Cassette

[0221] Banana (Musa spp. Cv. “Ladyfinger” AAA) embryogenic cellsuspensions are targeted for microprojectile-based stabletransformation. Cells are bombarded with pTEST3, as previouslydescribed, except the plasmid is co-transformed with 1 ug of pDHKAN(Pietrzak et al., 1986). This plasmid contains a CaMV 35S pro-NPTII-CaMV35S ter cassette, from which expression of the NPTII gene confersresistance to the antibiotics kanamycin or geneticin. Selection,culturing and regeneration of transgenic banana plants are doneessentially as described by Becker et al. (2000). Independent transgenicplants are confirmed to contain both the NPTII and uidA genes by PCR,using primer pairs NPT-F/NPT-R and uidA4/uidA5, respectively. Tenindependent transformants are selected for further studies.

[0222] Primers: NPT-F 5′-ATGATTGAACAAGATGGATT-3′ [SEQ ID NO:36] NPT-R5′-TGAGAAGAACTCGTCAAGA-3′ [SEQ ID NO:37] uidA45′-GTTATTGCCGGGAAAAGTGTACGTA- [SEQ ID NO:38] 3′ uidA55′-CTAGCTTGTTTGCCTCCCTGCTGCG- [SEQ ID NO:39] 3′

[0223] Tobacco (Nicotiana tabacum cv. “Samsun”) is transformed byAgrobacterium-mediated infection of leaf discs according to the methodof Horsch et al. (1988). Ten independent transgenic plants aretransformed with T-DNA from the plasmid pTEST4. Each line is shown tocontain the NPTII and uidA coding regions by PCR, as described above.

[0224] Leaf pieces from each of the ten transgenic banana and tobaccolines transformed with the Rep-activatable GUS expression cassette (i.e.pTEST3 [FIG. 10] and pTEST4 [FIG. 11], respectively) are bombarded withthe plasmid p35S-Rep. Three days post-bombardment, leaf pieces aresubjected to GUS histochemical assays. No GUS expression is evident inunshot leaf pieces from each of the ten banana and tobacco lines. Leafpieces, bombarded with the plasmid p35S-Rep, display multiple blueGUS-staining foci. Rep-directed nicking, joining, and replication of theTYDV-based MPEs is confirmed in these leaf pieces, as describedpreviously. These results indicate the TYDV Rep is capable of activatingGUS expression from a stably integrated copy of either plasmid, and ableto nick, join and replicate the TYDV-based MPE in vivo.

EXAMPLE 5

[0225] TYDV-Infection Activated Expression in Transgenic Tobacco

[0226] Each of the 10 transgenic tobacco lines is infiltrated withAgrobacterium cultures transformed with pBI-TYDV1.1 mer (refer toExample 2(f), FIG. 12) using the method of Boulton (1995). Over a twomonth period, samples are taken from the point of infection andthroughout the plant, and GUS expression assessed using histochemicalassays. GUS activity (i.e. blue staining tissue) is only noted inTYDV-infected plants, compared to mock-inoculated controls. Over time,GUS expression spreads, via the vasculature, from the initial point ofinfection to various plant parts. Rep-directed nicking, joining andreplication of the GUS expression cassette is established as previouslydescribed. This result suggests that TYDV infection is sufficient forreplicative release of the GUS expression cassette from an integratedchromosomal copy.

EXAMPLE 6

[0227] Transient Cell-Death Assays using Expression Vectors Based onBBTV

[0228] To demonstrate that barnase was capable of causing cell death,assays were carried out with the expression vectors (pTBN, pTBN6, pTBN1,FIGS. 1, 2 and 3). Negative controls were pUBN6 and pUBN1 (refer toExample 1, above). Each of these constructs was co-bombarded with a GUSvector into banana (Musa spp cv. Bluggoe) embryogenic cell suspensionsessentially as described by Dugdale et al., 1998. The GUS vectorcontained a strong promoter (maize Ubil, CaMV 35S or banana Act1 drivingthe expression of the reporter gene β-glucuronidase (Jefferson, 1987).Subsequent MUG assays for GUS activity showed that cells which weretransformed with either pTBN, pTBN6 or pTBN1 had lower GUS activity thandid the negative control (pUBN1 or pUBN6) (FIG. 14). This suggests thatintron splicing is still occurring, and at least in the case of pTBN1,does not differ significantly from the original pTBN vector. Thus, theinclusion of the BBTV replication elements into the intron did notsignificantly decrease the splicing efficiency or subsequent activity ofbarnase, relative to pTBN.

[0229] Experiments were conducted that showed that recircularization andreplication of BBTV based “1.1 mers” occurs in the presence of the Rep(gene product from BBTV DNA-1). It was also found that replication wasenhanced by inclusion of the gene product of BBTV DNA-S (a putativeretinoblastoma binding-like protein). Consequently, each of therecircularization vectors (pRTBN6 [FIG. 4], pRTBN1 [FIG. 5], pRUBN6,pRUBN1) was bombarded with BBTV DNA-1 and 5 “1.1 mers” and a GUSexpression vector. TABLE 2 Plasmid combinations used for microprojectilebombardment of banana cells x-axis label (FIG. 15) RTBN6 RUBN6 RTBN1RUBN1 BBTV1 ✓ ✓ ✓ ✓ BBTV5 ✓ ✓ ✓ ✓ GUS ✓ ✓ ✓ ✓ RTBN6 ✓ — — — RUBN6 — ✓ —— RTBN1 — — ✓ — RUBN1 — — — ✓

[0230] The results shown in FIG. 15 supported the previous expressionvector cell death assays (FIG. 14). Again, the constructs containinguntranslatable barnase (pRUBN6 and pRUBN1) had higher GUS activity thanthe translatable constructs.

[0231] Experiments were conducted to demonstrate that barnase activitywas induced only in the presence of the Rep protein. Consequently,assays were carried out ± the Rep (BBTV DNA-1 “1.1 mer”) to demonstratethat expression would only occur when it was present (Table 3).“Stuffer” DNA was used to keep a constant DNA concentration TABLE 3Plasmid combinations for microprojectile bombardment of banana cellsX-axis label (FIG. 16) RTBN6+ RTBN6− RUBN6+ RTBN1+ RTBN1− RUBN1+ BBTV1 ✓— ✓ ✓ — ✓ BBTV5 ✓ ✓ ✓ ✓ ✓ ✓ GUS ✓ ✓ ✓ ✓ ✓ ✓ RTBN6 ✓ ✓ — — — — RUBN6 — —✓ — — — RTBN1 — — — ✓ ✓ — RUBN1 — — — — — ✓ Stuffer — ✓ — — ✓ —

[0232] To observe if the recircularization constructs were able toreplicate in the presence of BBTV DNA-1 and 5 “1.1 mers”, untranslatableconstructs were included in transient banana cell replication assays.Cells were harvested at 0, 4 and 8 days after bombardment, totalcellular DNA extracted and analyzed using Southern hybridization.

[0233] Initially, the membranes were probed with a DIG-labelled CaMV 35Sprobe. No replicative forms were evident in cells at day 4 or 8.However, at day 0, potential replicative forms were present in very lowconcentrations in both pRUBN6 and pURBN1. The 35S probe was stripped andthe membranes reprobed with a DIG-labelled BBTV DNA-1 probe. High levelsof replication were observed in cells harvested on both day 4 and day 8,and almost none in cells harvested on day 0.

EXAMPLE 7

[0234] Cell-Death Assays using Expression Vectors Based on TYDV

[0235] (a) A Rep-Activatable Suicide Gene Vector to Confer Resistance toTYDV

[0236] The plasmid pRTBN (DNA Plant Technologies, Oakland, Calif.).contains the barnase coding region (339 bp) within which has beenincorporated the potato ST LS1 intron (188 bp). The entire barnase geneand intron was amplified from pRTBN by PCR using primers BARN.EXP1 andBARN.EX2. An untranslatable gene control was similarly amplified usingprimers BARN.UTR and BARN.EXP2.

[0237] Primers: BARN.EXP1 5′-GGATCCATGGCACAGGTTATCAACACGTTTGACG-3′ [SEQID NO: 40] BARN.EXP2 5′-CTAGAGTTATCTGATTTTTGTAAAGGTC-3′ [SEQ ID NO: 41]BARN.UTR 5′-GGATCCGCACAGGTTATCAACACGTTTGACG-3′ [SEQ ID NO: 42]

[0238] PCR products were cloned into pGEM-T vector. These clones weredesignated pGEM-BTR and pGEM-BUTR, respectively. The TYDV LIR (LIR-X)was excised as an EcoRI fragment from pGEM-T and inserted into the MfeIsite located within the potato LTS intron of pGEM-BTR and pGEM-BUTR.These plasmids were designated pBTR-LIR and pBUTR-LIR, respectively. TheLIR-containing barnase genes in pBTR-LIR and pBUTR-LIR were excised asBamHI/PstI fragments and inserted into similarly-digested pGUS2 vectorto replace the original uidA coding region. Plasmid pGUS2 contains aCaMV35S pro (530 bp)-uidA gene-CaMV 35S ter (200 bp). These constructswere designated p35S-BTR-LIR and p35S-BUTR-LIR, respectively (FIG. 17).Two control plasmids were constructed by excision of the barnase genesfrom pGEM-BTR and pGEM-BUTR with BamHI/PstI, and insertion intosimilarly digested pGUS2. These control plasmids were designatedp35S-BTR and p35S-BUTR, respectively (FIG. 18).

[0239] (b) Transient Assessment of TYDV Rep-Activated Barnase Activityin Monocotyledonous and Dicotyledonous Cells

[0240] In order to determine barnase activity in vivo, suicideconstructs are co-bombarded with a green fluorescent protein (gfp)expression cassette. Barnase expression and action (i.e. cell death) isconsidered to occur when a significant reduction in green fluorescentfoci is observed in comparison to the untranslatable barnase controls.

[0241] Banana (Musa spp. Cv. “Ladyfinger”) and tobacco (Nicotianatabacum NT-1) cells are bombarded with plasmids (i) p35S-BTR, (ii)p35S-BUTR, (iii) p35S-BTR-LIR, and (iv) p35S-BUTR-LIR (FIGS. 17 and 18)as described in Example 3, above. Each plasmid is co-bombarded with 1 ugof pWORM. The construct pWORM contains a CaMV 35S pro (530 bp)-gfp (750bp)-CaMV 35S ter (200 bp) cassette and has previously been shown toprovide strong green fluorescence in transient assays with both celltypes (Dugdale et al., 1998).

[0242] Three days post-bombardment, green fluorescence is visualisedusing a Leica MZ12 stereo microscope with GFP-Plus fluorescence module(excitation=490, emission=510). Both p35S-BTR and p35S-BTR-LIRsignificantly reduce gfp expression from pWORM (as determined by thenumber and intensity of green fluorescent foci) in comparison top35S-BUTR and p35S-BUTR-LIR. This result suggests that insertion of theTYDV LIR into the ST LS1 intron within p35S-BTR does not interfere withintron processing nor inhibit bamase expression.

[0243] (c) Construction of the TYDV Rep-Activatable Barnase Vector

[0244] The CaMV 35S promoter, barnase 5′ gene half, ST LS5′ intron halfand TYDV LIR are re-amplified from p35S-BTR-LIR (FIG. 17A) by PCR usingprimers 35S-IE (SEQ ID NO:21) and LIR-R (SEQ ID NO:20). The PCR productis cloned into pGEM-T vector and sequence-verified. This plasmid isdesignated pGEMB5′. The TYDV LIR, ST LS1 3′ intron half, bamase 3′ genehalf and nos terminator is excised from p35S-BTR-LIR as a XhoI/SacIfragment, the TYDV SIR is excised from pGEM-SIR as a SacI/NcoI fragment,and the CaMV 35S promoter, barnase 5′ gene half, ST LS1 5′ intron halfand TYDV LIR are excised from pGEMB5′ as a NcoI/SacII partial fragment.Inserts are ligated together with XhoI/SacII digested pBluescript II(Stratagene). The resulting construct is designated pBTR.test1 (FIG.19A). The untranslatable control vector is similarly prepared and theresulting construct designated pBUTR.test1 (FIG. 19B).

[0245] (d) Transient TYDV Rep-Activated Barnase Expression in Monocotand Dicot Cells.

[0246] Banana (Musa spp. Cv. “Ladyfinger”) and tobacco (Nicotianatabacum NT-1) cells are bombarded, as described above, with the plasmidcombinations listed in Table 4,below:— TABLE 4 Plasmid combinations fortransient transformation assays in banana and tobacco cells, and thereresulting gfp expression (asssessed as + or −) pBTR- pBUTR- p35S- pBTR-pBUTR- test1 test1 Plasmid BTR p35S-BUTR test1 test1 pWORM pWORMcombination pWORM pWORM pWORM pWORM p35S-Rep p35S-Rep Green no yes yesyes no yes fluorescent foci 3 days post- bombard- ment

[0247] Results in Table 4 suggest that barnase expression (and thereforecell death) is only activated from pBTR-test1 when the TYDV Rep issupplied in trans. Rep-activated expression of the untranslatablebarnase gene cassette (pBUTR-test1) results in no significant reductionin gfp expression from pWORM. Rep-assisted nicking, joining, andreplication of the MPEs from cells bombarded with pBUTR-test1, pWORM,and p35S-Rep is confirmed as previously described, except primersBARN.UTR (SEQ ID NO:40) and BARN.EXP2 (SEQ ID NO:41) are used for PCRand a DIG-labelled barnase-specific probe is synthesised using thebefore-mentioned primers.

[0248] (e) Construction of Binary Plasmids Containing theRep-Activatable Barnase Gene Cassettes

[0249] Rep-activatable barnase cassettes are excised from pBTR-test1 andpBUTR-test1 as PvuII fragments and inserted into the unique EcoRI site(blunt ended using DNA polymerase I large Klenow fragment) locateddownstream of the CaMV 35S pro-NPTII-CaMV 35S ter cassette in the binaryplasmid pTAB5 (CSIRO, Canberra, Australia). The resulting constructs aredesignated pTAB-BTR1 and pTAB-BUTR1, respectively. Both vectors areintroduced into Agrobacterium tumefaciens (LBA4404) by electroporationusing the method of Singh et al. (1993).

[0250] (f) Stable Transformation of a Monocotyledonous and aDicotyledonous Plant with the Rep-Activatable Barnase Cassettes.

[0251] Stable transformation of banana (Musa spp. Cv. “Ladyfinger”) andtobacco (Nicotiana tabacum cv. “Samsun”) is done as described in Example4, above, except plasmids pBTR-test1 and pBUTR-test1 are independentlyco-transformed with pDHKAN for stable banana transformation andAgrobacterium cultures harbouring the plasmids pTAB-BTR1 and pTAB-BUTR1are used for Agrobacterium-mediated transformation of tobacco leafdisks.

[0252] Transformed plants are confirmed to contain the barnase genecassettes and the NPTII gene by PCR using primer pairs LIR-F/LIR-R (SEQID NO:19/SEQ ID NO:20) and NPT-F/NPT-R (SEQ ID NO:36/SEQ ID NO:37),respectively. Ten independent transgenic lines of both monocotyl-edonousand dicotyledonous species are selected for further studies.

[0253] (g) Rep-Activated Hypersensitive Resistance to TYDV in TransgenicTobacco

[0254] Rep-activation of bamase expression in the ten independenttobacco plants transformed with pTAB-BTR1 and pTAB-BUTR1 is initiallytested by particle bombardment of the p35S-Rep construct into leafpieces, as described. Two days post-bombardment, necrosis of bombardedareas is only evident on leaves of tobacco plants transformed with theRep-activatable translatable barnase gene cassette (pTAB-BTR1) incomparison to the untranslatable control (pTAB-BUTR-1). This resultsuggests introduction of the TYDV Rep in trans is sufficient forreplicative release of the barnase expression cassette from anintegrated chromosomal copy. Rep-assisted nicking, joining, andreplication is confirmed in leaf pieces of tobacco plants transformedwith pTAB-BUTR1, as was described for the transient assays in NT-1cells.

[0255] To demonstrate hypersensitive resistance to TYDV in transgenictobacco,. viruliferous leafhoppers (Orosius argentatus) are allowed tofeed on plants for up to 2 days. Over the following week, plants areinspected for characteristic TYDV symptoms as described originally byHill (1937). Plants transformed with the Rep-activatable, untranslatablebarnase expression cassette (pTAB-BUTR1) produce typical TYDV symptoms,including dwarfing, yellowing, bending down of margins and tips of youngleaves, and shortening of the internodes. In contrast, tobacco plantstransformed with the Rep-activatable, translatable barnase expressioncassette (pTAB-BTR1) display atypical necrotic lesions at the site ofaphid feeding (most likely the result of barnase-induced cell death).These plants develop normally over the ensuing three months, incomparison to uninfected tobacco plants, and at no point developsymptoms characteristic of TYDV infection.

[0256] Total gDNA is isolated from leaves of each of the 20 transgenictobacco plants and uninfected controls, two weeks post-infection, usingthe method of Stewart and Via (1993). Total gDNA (1 μg) is used as atemplate for a PCR with primers designed to the TYDV coat protein gene(CP-F and CP-R). The 765 bp coat protein gene, and therefore virusgenome, is only detected in tobacco transformed with pTAB-BUTR1. Thisresult suggests that tobacco plants transformed with pTAB-BTR1 areresistant to TYDV infection and remain free of TYDV-induced symptomsover extended periods of time.

[0257] Primers: CP-F 5′-ATGGCGGGCCGGTATAAGGGTTTGG-3′ [SEQ ID NO: 43]CP-R 5′-TTATTGATTGCCAACTGATTTGAAAT-3′ [SEQ ID NO: 44]

EXAMPLE 8

[0258] gfp Vector Constructions—Based on BBTV

[0259] A similar series of vectors, based on the Rep-activatable barnasecassettes, were 5 constructed using the reporter gene encoding greenfluorescent protein (GFP) and BBTV intergenic regions. Both expressionand re-circularization vectors were constructed by overlapping PCR in amanner similar to that of the pRTBN series of vectors and cloned into apUC19 vector. Primers used in the construction of these vectors areindicated in Table 5. In some cases plasmids pBN, pTBN6 and pTBN1 wereused as templates for PCRs. TABLE 5 Oligonucleotide sequences PrimerName Sequence (5′-3′) +1 35SH AAGCTTCATGGAGTCAAAGA (SEQ ID NO: 45) +25′mGFPBam GGATCCATGAGTAAAGGAGAAGAACTT (SEQ ID NO: 46) +3 GFPB1AAGTCAAGTTTGAGGTAAGTTTCTGCTTC (SEQ ID NO: 47) −3 B2GFPGAAGCAGAAACTTACCTCAAACTTGACTT (SEQ ID NO: 448) +4 B1GFPTTGTTGATGTGCAGGGAGACACCCTCGTC (SEQ ID NO: 49) −4 GFPB2GACGAGGGTGTCTCCCTGCACATCAACAA (SEQ ID NO: 50) −5 3′mGFPSacGAGCTCTTATTTGTATAGTTCATCCAT (SEQ ID NO: 51) −6 NOS4-HAAGCTTTTCGCCATTCAGGCTGC (SEQ ID NO: 52) +7 B3-6TATCATTAATTAGTAAGTTGTGCTGTAA (SEQ ID NO: 53) −8 B4-6AATATTATATTACTCGCTTCTGCCTTCC (SEQ ID NO: 54) +9 B5-IATCATTAATTAGTCACACTATGACAAAAG (SEQ ID NO: 55) −10  B6-IAATATTATATTACTGATGCAAATGTCCCCG (SEQ ID NO: 56)

[0260] Initially, plasmids pGI (SEQ ID NO:69), pGI6 (SEQ ID NO:70), pGI1(SEQ ID NO:71) were constructed and are shown in FIGS. 20, 21 and 22,respectively. Ultimately, plasmids pRGI6, pRGI1, pRGI1/6 wereconstructed and are shown in FIGS. 23, 24 and 25, respectively. In FIGS.20 to 25, intron refers to the potato ST-LS1 intron, intergenic regionsare derived from either BBTV DNA-1 or -6, and CT and NT refer to theC-terminal and N-terminal portions of the GFP reporter gene.

[0261] The efficacy of these various constructs is assessed in transientassays via micro-projectile bombardment of banana cell suspensions. Thefirst transient assays, measure GFP expression to determine whether thevarious monomers are replicatively released and re-circularized, andthat the GFP gene is transcribed, processed and expressed correctly. ABBTV DNA-1 1.1 mer is mixed in equimolar amounts of either pGI1 or pGI6and bombarded into banana embryogenic cell suspensions. Eight dayspost-bombardment, replicative release and circularization are assayed bySouthern hybridization, using a GFP-specific probe. GFP expression ismonitored using a GFP microscope and quantified using Western blotanalysis and a GFP-specific antisera.

[0262] Southern hybridization indicates the presence of monomericcircular molecules from either pGI1 or pGI6P only when the BBTV DNA-11.1 mer is delivered in trans. Similarly, GFP expression is onlydetected, when the viral-derived 1.1 mer is present.

EXAMPLE 9

[0263] Stable Transformations of Banana for Barnase-Induced Resistanceto BBTV

[0264] Banana embryogenic cell suspensions of both Bluggoe and Cavendishare co-transformed with pRTBN6 and a plasmid carrying the selectablemarker gene using microprojectile bombardment (Becker et al., 2000).Regenerated plantlets are assayed by PCR to determine whether a completecopy of the Rep-activatable barnase cassette has been incorporated intothe banana genome. Positive transformants are multiplied and, initiallyfive plants from each transformation are challenged with 20 BBTVviruliferous aphids. Southern hybridization analyses are used to comparelevels of viral DNA accumulation between transformed and non-transformedplants. Promising transgenic lines are further multiplied, re-challengedand assayed.

[0265] In nearly all cases, transgenic banana plants show no evidence ofbanana bunchy top disease, in comparison to controls. Rather, atypicalnecrosis at the point of aphid feeding is observed, most likelyreflecting barnase-induced cell death. Using PCR and Southernhybridisation the coat protein gene of BBTV can not be detected in anyplant part tested, suggesting these lines are resistant to BBTVinfection, replication and spread.

EXAMPLE 10

[0266] Tissue-Specific and Inducible Rep-Activated Expression-Based onTYDV

[0267] In order to control the site of Rep expression, and thereforetransgene activation/replication in planta, tissue-specific promoterswere employed.

[0268] (a) Constitutive Expression

[0269] The vector pTAB16 contains a CaMV 35S pro-bar selection gene-ocster and CaMV 35S pro-uidA-CaMV 35S ter cassettes located between theright and left T-DNA borders in pBIN16. The CaMV 35S-TYDV Rep genecassette is excised from p35S-Rep by EcoRI/BamHI digestion and insertedinto similarly digested pTAB16 vector to replace the original CaMV35S-uidA cassette. This construct is designated pTAB-TYDV-Rep.

[0270] (b) Seed-Specific Expression

[0271] The 1 kb rice glutelin promoter (Genbank Accession X52153) hasbeen shown to direct seed-specific reporter gene expression in tobacco(Leisy et al., 1989). The rice glutelin promoter is excised from theplasmid pGT3-JEFLK (Miller, 2001) by NcoI digestion and ligated intosimilarly digested pTAB-TYDV-Rep vector to replace the original CaMV 35Spromoter. This construct is designated pGL-TYDV-Rep.

[0272] (c) Root-Specific Expression

[0273] The 880 bp Arabidopsis thaliana root-specific kinase homolog(ARSK1) promoter (Genbank Accession L22302) has been shown to directtissue-specific uidA reporter gene expression in epidermal,endoepidermal, and cortex regions of A. thaliana roots (Hwang andGoodman, 1995). The ARSK1 promoter is amplified from A. thaliaia gDNA byPCR using primers ARSK-F and ARSK-R. The resulting PCR product is clonedinto pGEM-T and the sequence verified. The ARSK1 promoter is excisedfrom pGEM-T by NcoI digestion and ligated into similarly-digestedpTAB-TYDV-Rep to replace the original CaMV 35S promoter. This constructis designated pAR-TYDV-Rep.

[0274] Primers: ARSK-F 5′-CCATGGATCTCATTCTCCTTCAACAAGGCG-3′ [SEQ ID NO:57] ARSK-R 5′-CCATGGTTTCAACTTCTTCTTTTGTGTTATTTG-3′ [SEQ ID NO: 58]

[0275] (d) Wound-Inducible Expression

[0276] The 2032 bp Asparagus officinalis PR gene (AoPR1) promoter(Genbank Accession: A26573) has been shown to direct strong reportergene expression in wounded and actively dividing cell types such as, forexample, callus (Firek et al., 1993). The AoP1 promoter is amplifiedfrom A. officinalis gDNA by PCR using primers AoPR-F and AoPR-R. Theresulting PCR product is cloned into pGEM-T and the sequence verified.The AoPR1 promoter is excised from pGEM-T by NcoI digestion and ligatedinto similarly digested pTAB-TYDV-Rep to replace the original CaMV 35Spromoter. This construct is designated pAo-TYDV-Rep.

[0277] Primers: AoPR-F 5′-GAATTCAGGGGTAAGTTTGCAAATATC-3′ [SEQ ID NO: 59]AoPR-R 5′-CGAGGTTGTGCCAGTCGAGCATTGCC-3′ [SEQ ID NO: 60]

[0278] (e) Alcohol-Inducible Expression

[0279] The ALC switch, derived from Aspergillus nidulans, is analcohol-inducible promoter system based on the AlcA promoter and AlcRreceptor. The ALC switch has been shown to function in plant systemsusing a uidA reporter gene model (Caddick et al., 1998). The plasmidpSRNAGS (BTI, Cornell University, Ithaca, N.Y.) contains a CaMV 35Spro-AlcR gene-nos ter and AlcA pro-uidA reporter gene-CaMV 35S tercassette in pBIN16. The CaMV 35S pro-AlcR gene-nos ter-AlcA pro cassetteis amplified from pSRNAGS by PCR using primers 35S-IE (SEQ ID NO:21) andAlc-R (SEQ ID NO:61). The PCR product is cloned into pGEM-T and sequenceverified. The insert is then excised by NcoI digestion and ligated intosimilarly-digested pTAB-TYDV-Rep to replace the original CaMV 35Spromoter. This construct is designated pAlc-TYDV-Rep.

[0280] Primer: Alc-R 5′-CCATGGTTTGAGGCGAGGTGATAGGATTGG-3′ [SEQ ID NO:61]

[0281] Each of the binary TYDV Rep-containing plasmids are introducedinto Agrobacterium tumefaciens (LBA4404) by electroporation using themethod of Singh et al. (1993).

[0282] (f) Tissue-Specific and Inducible Rep Expression Directs ReporterGene Activation in Precise Tissue Types

[0283] Leaves from tobacco plants transformed with the plasmid pTEST4are super-infected with Agrobacterium harbouring the plasmidspTAB-TYDV-Rep, pGL-TYDV-Rep, pAR-TYDV-Rep, pAo-TYDV-Rep, andpAIc-TYDV-Rep using the method of Horsch et al. (1988). In this case,selection of transformed tobacco plants is achieved using the herbicidephosphoinothricin ammonium (PPT). Transgenic plants are confirmed tocontain the TYDV Rep gene by PCR using the primers TYDV.RepF andTYDV.RepR. Ten independent transformants for each plasmid are selectedfor further studies. Plants are grown to maturity, allowed to flower andseed collected. Different plant organs from independent transformants,including leaves, stems, roots, flower, and seed, are collected and GUSactivity detected using histochemical assays.

[0284] Tobacco plants super-transformed with the plasmid pTAB-TYDV-Repdisplay strong GUS expression throughout all plant parts tested. Thelevel of GUS expression in these plants is considerably higher thanplants transformed with the non-replicating control, pTAB 16.

[0285] In contrast, plants super-transformed with the plasmidpGL-TYDV-Rep show strong GUS expression in the seeds only, plantssuper-transformed with the plasmid pAR-TYDV-Rep show strong GUSexpression in the roots only, plants super-transformed with the plasmidpAo-TYDV-Rep show strong GUS expression in wounded and meristematiccells, and plants super-transformed with the plasmid pAlc-TYDV-Rep showstrong constitutive GUS expression when drenched in a 1% v/v ethanolsolution only.

[0286] Rep-assisted nicking, joining and replication of the GUSexpression cassette is confirmed (as described previously) in all tissuetypes of tobacco plants super-transformed with the plasmidpTAB-TYDV-Rep. In contrast, this activity is only detected in the seedsof plants super-transformed with the plasmid pGL-TYDV-Rep, in the rootsof plants super-transformed with the plasmid pAR-TYDV-Rep, at the siteof wounding in plants super-transformed with the plasmid pAo-TYDV-Rep,and constitutively in ethanol-induced plants super-transformed with theplasmid pAlc-TYDV-Rep. This result suggests tissue-specific or inducedexpression of the TYDV Rep gene confers high-level expression from theRep-activatable GUS cassette in those tissue types only.

EXAMPLE 11

[0287] TMV p50 and hrmA Gene Mediated Resistance—Based on TYDV

[0288] (a) TYDV Rep-Activated Expression of the TMV p50 HelicaseFragment Induces Systemic Acquired Resistance (SAR) in Tobacco

[0289] Previous studies (Erickson et al., 1999) have demonstrated thatnon-viral expression of the 50 kDa tobacco mosaic virus (TMV) helicasefragment (p50) is sufficient to induce the N-mediated hypersensitiveresponse (HR) in suitable tobacco varieties (e.g. Nicotiana tabacuin cv.“Petite Havana” SR1 homozygous for the N gene). The defence response ischaracterised by cell death at the site of virus infection and inductionof the systemic acquired resistance (SAR) pathway with resultinginhibition of viral replication and movement.

[0290] The p50 gene fragment is amplified by PCR using primers p50-F andp50-R from cloned genomic TMV DNA (Plant Gene Expression Centre,University of California, USA). The PCR product is cloned into pGEM-Tand the sequence verified. The catalase intron containing the TYDV LIRfrom pTEST3 (FIG. 10) is engineered in frame into the unique EcoRI sitein the p50 coding region. This plasmid is designated pGEM50-LIR. ApUC-based Rep-activatable p50 gene plasmid is subsequently constructed,as described for pTEST3 in Example 2, above. The cassette is insertedinto pART27, as was described for pTEST4. The resulting p50Rep-activatable binary plasmid is designated pSAR1. The plasmid pSAR1 isused to transform Agrobacterium as previously described.

[0291] Primers: p50-F 5′-CCATGGAGATAGAGTCTTTAGAGCAGTTTC-3′ [SEQ ID NO:62] p50-R 5′-GGATCCTATTGTGTTCCTGCATCGACCTTA-3′ [SEQ ID NO: 63]

[0292] (b) Systemic Acquired Resistance to TYDV in Tobacco

[0293] Ten tobacco plants (Nicotiana tabacum cv. “Petite Havana” SR1homozygous for the N gene) transformed with T-DNA from plasmid pSAR1 areobtained by Agrobacterium-mediated transformation and confirmed tocontain the Rep-activatable cassette and NPTII gene by PCR as describedabove. Plants are infected with TYDV and observed for symptoms, aspreviously described. Tobacco plants transformed with theRep-activatable, p50 gene display atypical hypersensitive necrosis atthe site of aphid feeding, two days post infection. These plants developnormally over the ensuing 3 months, in comparison to infectednon-transgenic tobacco plants, which display typical TYDV-inducedsymptoms.

[0294] TYDV genomic DNA is detected in inoculated non-transgenic tobaccobut not in transgenic nor uninfected control lines, as previouslydescribed. This result suggests TYDV Rep-induced expression of the TMVp50 gene is sufficient to stimulate the N gene hypersensitive responsein suitable tobacco cultivars and provide resistance to TYDV infection.

[0295] (c) Wound-Inducible TYDV Rep Expression Activates TMV p50 GeneExpression and Triggers SAR to a Variety of Pathogens.

[0296] Leaves from pSAR1-transformed tobacco plants are super-infectedwith Agrobacterium containing the plasmid pAo-TYDV-Rep (Example 10), aspreviously described. Ten super-transformed lines, confirmed to containthe wound-inducible Rep gene, are selected for further studies.Transgenic plants and suitable controls are subjected to infection witha variety of viral pathogens e.g. (i) aphid transmission of tobacco veinmottling virus, (ii) tobacco rattle virus via the nematode vectorParatrichodorus pachydermus, and (iii) biolistic introduction of aninfectious BeYDV 1.1 mer. Over time, plants are observed forcharacteristic viral-induced symptoms. All transgenic plants displayatypical hypersensitive necrosis at the site of virus inoculation incomparison to controls.

[0297] (d) Wound-Inducible Expression of TYDV Rep ActivateshrmA-Mediated Broad Range Pathogen Resistance in Tobacco

[0298] The hrmA gene product from Pseudomonas syringae pv. syringae hasbeen shown to activate pathogen related genes in a number of tobaccocultivars, and confer resistance to variety of pathogens, includingviruses, fingi and bacteria (Shen et al., 2000).

[0299] The hrmA gene is amplified from a plasmid containing the codingsequence by PCR using primers him-F and hrm-R. A pUC-basedRep-activatable hrmA plasmid is subsequently constructed, as describedin Example 2, above, for pTEST3. The cassette is inserted into pART27,as described for pTEST4. The resulting hrmA-activatable binary plasmidis designated pSAR2. The plasmid pSAR2 is used to transformAgrobacterium, as described. hrm-F5′-CCATGGGCATGCACGCTTCTCCAGCGTAGAAGCG-3′ [SEQ ID NO: 64] hrm-R5′-GGATCCTCAGTTTCGCGCCCTGAGCGCCGG-3′ [SEQ ID NO: 65]

[0300] Ten tobacco plants (Nicotiana tabacum) transformed with T-DNAfrom plasmid pSAR2 are obtained by Agrobacterium-mediated transformationand confirmed to contain the Rep-activatable cassette and NPTII gene byPCR as previously described. Leaves from pSAR2 transformed tobaccoplants are super-infected with Agrobacterium containing the plasmidpAo-TYDV-Rep, as previously described. Ten super-transformed lines,confirmed to contain the wound-inducible Rep gene, are selected forfurther studies. Transgenic plants and suitable controls are subjectedto infection with a variety of pathogens e.g. tobacco vein mottlingvirus, tobacco etch virus, blank shank fungus Phytophthora parasitica,and wild fire bacterium Pseudomonas syrinagae pv. tabaci. Over time,plants are observed for characteristic pathogen-induced symptoms. Alltransgenic plants display atypical hypersensitive necrosis at the siteof inoculation, in comparison to controls.

EXAMPLE 12

[0301] Over Expression of Human Serum Albumin—Based on TYDV

[0302] (a) Rep-Activated, Over-Expression of a Commercially ImportantProtein, Human Serum Albumin

[0303] Albumin is a soluble, monomeric protein which comprises aboutone-half of the blood serum protein. Albumin functions primarily as acarrier protein for steroids, fatty acids, and thyroid hormones andplays a role in stabilizing extracellular fluid volume.

[0304] The 1831 bp Human serum albumin (HSA) coding region (Lawn et al.,1981) is amplified from a plasmid containing the gene by PCR usingprimers Alb-F and Alb-R. A pUC-based Rep-activatable HSA plasmid issubsequently constructed, as described in Example 2, above, for pTEST3.The cassette is inserted into pART27, as described for pTEST4. Theresulting HSA-activatable binary plasmid is designated pHSA1. Theplasmid pHSA1 is used to transform Agrobacterium, as described.

[0305] Ten tobacco plants (Nicotiana tabacuin) transformed with T-DNAfrom plasmid PHSA1 are obtained by Agrobacterium-mediated transformationand confirmed to contain the Rep-activatable cassette and NPTII gene byPCR, as described. Leaves from pHSA1 transformed tobacco plants aresuper-infected with Agrobacterium containing plasmids pTAB-TYDV-Rep,pGL-TYDV-Rep, pAo-TYDV-Rep, pAlc-TYDV-Rep, as described. Tensuper-transformed lines from each transformation are confirmed tocontain the Rep gene, and selected for further studies. Plants are grownto maturity and HSA content assessed using ELISA and antibodies raisedto the HSA gene product. HSA protein is detected at high levels but indifferent plant parts or under specific conditions; i.e. constitutively(pTAB-TYDV-Rep), seed only (pGL-TYDV-Rep), wounded tissues(pAo-TYDV-Rep) and constitutively in alcohol treated plants(pAlc-TYDV-Rep). A proposed model for Rep-activated expression of thehuman serum albumin gene from the plasmid pHSA1 is depicted in FIG. 26.

[0306] Primers: Alb-F 5′-CCATGGAGATGAAGTGGGTAACCTTTATTTCC-3′ [SEQ ID NO:72] Alb-R 5′-GGATCCTTATAAGCCTAAGGCAGCTTGACT-3′ [SEQ ID NO: 73]

[0307] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features.

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1 73 1 20 DNA Artificial Sequence Synthetic oligonucleotide 1 aagcttcatggagtcaaaga 20 2 29 DNA Artificial Sequence Synthetic oligonucleotide 2tcatttggag aggatccatg gcacaggtt 29 3 29 DNA Artificial SequenceSynthetic oligonucleotide 3 aacctgtgcc atggatcctc tccaaatga 29 4 29 DNAArtificial Sequence Synthetic oligonucleotide 4 aaaatcagat aagagctcgatcgttcaaa 29 5 29 DNA Artificial Sequence Synthetic oligonucleotide 5tttgaacgat cgagctctta tctgatttt 29 6 23 DNA Artificial SequenceSynthetic oligonucleotide 6 aagcttttcg ccattcaggc tgc 23 7 28 DNAArtificial Sequence Synthetic oligonucleotide 7 tatcattaat tagtaagttgtgctgtaa 28 8 28 DNA Artificial Sequence Synthetic oligonucleotide 8ttacagcaca acttactaat taatgata 28 9 28 DNA Artificial Sequence Syntheticoligonucleotide 9 ggaaggcaga agcgagtaat ataatatt 28 10 28 DNA ArtificialSequence Synthetic oligonucleotide 10 aatattatat tactcgcttc tgccttcc 2811 29 DNA Artificial Sequence Synthetic oligonucleotide 11 atcattaattagtcacacta tgacaaaag 29 12 29 DNA Artificial Sequence Syntheticoligonucleotide 12 ttgtcatagt gtgactaatt aatgataat 29 13 29 DNAArtificial Sequence Synthetic oligonucleotide 13 gacatttgca tcagtaatataatatttca 29 14 29 DNA Artificial Sequence Synthetic oligonucleotide 14aatattatat tactgatgca aatgtcccg 29 15 29 DNA Artificial SequenceSynthetic oligonucleotide 15 tcagataaga gctcagtaac agcaacaac 29 16 29DNA Artificial Sequence Synthetic oligonucleotide 16 gctgttactgagctcttatc tgatctttg 29 17 23 DNA Artificial Sequence Syntheticoligonucleotide 17 aagcttattt cccaaatata cgt 23 18 21 DNA ArtificialSequence Primer 18 ggatccgcac aggttatcaa c 21 19 41 DNA ArtificialSequence Primer 19 gctcttcctg caggcggccg cattaaggct caagtaccgt a 41 2041 DNA Artificial Sequence Primer 20 gctcttcgtc gacgaattca ttttcaactttgggatgtca c 41 21 24 DNA Artificial Sequence Primer 21 gaattccatggagtcaaaga ttca 24 22 32 DNA Artificial Sequence Primer 22 gcccgctgcagagtttaaag aaagatcaaa gc 32 23 34 DNA Artificial Sequence Primer 23gccccggtcg acgatctatt ttttaattga ttgg 34 24 33 DNA Artificial SequencePrimer 24 ttcgagctgg tcacctgtaa ttcacacgtg gtg 33 25 18 DNA ArtificialSequence Primer 25 ccatggtaga tctgaggg 18 26 25 DNA Artificial SequencePrimer 26 tacgtacact tttcccggca ataac 25 27 23 DNA Artificial SequencePrimer 27 gtaacgcgct ttcccaccaa cgc 23 28 26 DNA Artificial SequencePrimer 28 ctcgagatta aggctcaagt accgta 26 29 21 DNA Artificial SequencePrimer 29 agtttaaaga aagatcaaag c 21 30 20 DNA Artificial SequencePrimer 30 aattaaccct cactaaaggg 20 31 32 DNA Artificial Sequence Primer31 gcatgcaaga gttggcggta gattccgcat gt 32 32 35 DNA Artificial SequencePrimer 32 gctcttcgcg gccgcgctcc tgaatcgtcg agtca 35 33 22 DNA ArtificialSequence Primer 33 tttaaacgtt taggggttag ca 22 34 18 DNA ArtificialSequence Primer 34 tcagtgactc gacgattc 18 35 18 DNA Artificial SequencePrimer 35 ttaatatgcc ttcagccc 18 36 20 DNA Artificial Sequence Primer 36atgattgaac aagatggatt 20 37 18 DNA Artificial Sequence Primer 37tgagaagaac tcgtcaag 18 38 25 DNA Artificial Sequence Primer 38gttattgccg ggaaaagtgt acgta 25 39 25 DNA Artificial Sequence Primer 39ctagcttgtt tgcctccctg ctgcg 25 40 34 DNA Artificial Sequence Primer 40ggatccatgg cacaggttat caacacgttt gacg 34 41 28 DNA Artificial SequencePrimer 41 ctagagttat ctgatttttg taaaggtc 28 42 31 DNA ArtificialSequence Primer 42 ggatccgcac aggttatcaa cacgtttgac g 31 43 25 DNAArtificial Sequence Primer 43 atggcgggcc ggtataaggg tttgg 25 44 26 DNAArtificial Sequence Primer 44 ttattgattg ccaactgatt tgaaat 26 45 20 DNAArtificial Sequence Synthetic oligonucleotide 45 aagcttcatg gagtcaaaga20 46 27 DNA Artificial Sequence Synthetic oligonucleotide 46 ggatccatgagtaaaggaga agaactt 27 47 29 DNA Artificial Sequence Syntheticoligonucleotide 47 aagtcaagtt tgaggtaagt ttctgcttc 29 48 29 DNAArtificial Sequence Synthetic oligonucleotide 48 gaagcagaaa cttacctcaaacttgactt 29 49 29 DNA Artificial Sequence Synthetic oligonucleotide 49ttgttgatgt gcagggagac accctcgtc 29 50 29 DNA Artificial SequenceSynthetic oligonucleotide 50 gacgagggtg tctccctgca catcaacaa 29 51 27DNA Artificial Sequence Synthetic oligonucleotide 51 gagctcttatttgtatagtt catccat 27 52 23 DNA Artificial Sequence Syntheticoligonucleotide 52 aagcttttcg ccattcaggc tgc 23 53 28 DNA ArtificialSequence Synthetic oligonucleotide 53 tatcattaat tagtaagttg tgctgtaa 2854 28 DNA Artificial Sequence Synthetic oligonucleotide 54 aatattatattactcgcttc tgccttcc 28 55 29 DNA Artificial Sequence Syntheticoligonucleotide 55 atcattaatt agtcacacta tgacaaaag 29 56 30 DNAArtificial Sequence Synthetic oligonucleotide 56 aatattatat tactgatgcaaatgtccccg 30 57 30 DNA Artificial Sequence Primer 57 ccatggatctcattctcctt caacaaggcg 30 58 33 DNA Artificial Sequence Primer 58ccatggtttc aacttcttct tttgtgttat ttg 33 59 27 DNA Artificial SequencePrimer 59 gaattcaggg gtaagtttgc aaatatc 27 60 26 DNA Artificial SequencePrimer 60 cgaggttgtg ccagtcgagc attgcc 26 61 30 DNA Artificial SequencePrimer 61 ccatggtttg aggcgaggtg ataggattgg 30 62 30 DNA ArtificialSequence Primer 62 ccatggagat agagtcttta gagcagtttc 30 63 30 DNAArtificial Sequence Primer 63 ggatcctatt gtgttcctgc atcgacctta 30 64 34DNA Artificial Sequence Primer 64 ccatgggcat gcacgcttct ccagcgtaga agcg34 65 30 DNA Artificial Sequence Primer 65 ggatcctcag tttcgcgccctgagcgccgg 30 66 1294 DNA Artificial Sequence Synthetic construct 66catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 60gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 120gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 300ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 360acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 480gcaagaccct tcctctatat aaggaagttc atttcatttg gagaggatcc catggcacag 540gttatcaaca cgtttgacgg ggttgcggat tatcttcaga catatcataa gctacctgat 600aattacatta caaaatcaga agcacaagcc ctcggctggg tggcatcaaa agggaacctt 660gcagacgtcg ctccggggaa aagcatcggc ggagacatct tctcgaacag gtaagtttct 720gcttctacct ttgatatata tataataatt atcattaatt agtagtaata taatatttca 780aatatttttt tcaaaataaa agaatgtagt atatagcaat tgcttttctg tagtttataa 840gtgtgtatat tttaatttat aacttttcta atatatgacc aaaatttgtt gatgtgcagg 900gagggcaagc tcccgggcaa aagcggacga acatggcgtg aagcggatat taactataca 960tcaggcttca gaaattcaga ccggattctt tactcaagcg actggctgat ttacaaaaca 1020acggaccatt atcagacctt tacaaaaatc agataactct agagtttctt aagattgaat 1080cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt taagcatgta 1140ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat tagagtcccg 1200caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta ggataaatta 1260tcgcgcgcgg tgtcatctat gttactagat cggg 1294 67 2708 DNA ArtificialSequence Synthetic construct 67 gcgaagttgt gctgtaatgt taattaataaaacgtatatt tgggaaattg atagttgtat 60 aaaacataca acacactatg aaatacaagacgctatgaca aatgtacggg tatctgaatg 120 agttttagta tcgcttaagg gccgcaggcccgttaaaaat aataatcgaa ttataaacgt 180 tagataataa tcagagatag gtgatcagataatataaaca taaacgaagt atatgccggt 240 acaataataa aataagtaat aacaaaaaaaatatgtatac taatctctga ttggttcagg 300 agaaaggccc accaactaaa aggtggggagaatgtcccga tgacgtaagc acgggggact 360 attattaccc cccgtgctcg ggacgggacatgacgtcagc aaggattata atgggctttt 420 tattagccca tttattgaat tgggccgggttttgtcattt tacaaaagcc cggtccagga 480 taagtataat gtcacgtgcc gaattaaaaggttgcttcgc cacgaagaaa cctaatttga 540 ggttgcgtat tcaatacgct accgaatatctattaatatg tgagtctctg ccgaaaaaaa 600 tcagagcgaa agcggaaggc agaagcgagtaatataatat ttcaaatatt tttttcaaaa 660 taaaagaatg tagtatatag caattgcttttctgtagttt ataagtgtgt atattttaat 720 ttataacttt tctaatatat gaccaaaatttgttgatgtg cagggagggc aagctcccgg 780 gcaaaagcgg acgaacatgg cgtgaagcggatattaacta tacatcaggc ttcagaaatt 840 cagaccggat tctttactca agcgactggctgatttacaa aacaacggac cattatcaga 900 cctttacaaa aatcagataa gagctcgtttcttaagattg aatcctgttg ccggtcttgc 960 gatgattatc atataatttc tgttgaattacgttaagcat gtaataatta acatgtaatg 1020 catgacgtta tttatgagat gggtttttatgattagagtc ccgcaattat acatttaata 1080 cgcgatagaa aacaaaatat agcgcgcaaactaggataaa ttatcgcgcg cggtgtcatc 1140 tatgttacta gatcggggaa ttcactggccgtcgttttac aacgtcgtga ctgggaaaac 1200 cctggcgtta cccaacttaa tcgccttgcagcacatcccc ctttcgccag ctggcgtaat 1260 agcgaagagg cccgcaccga tcgcccttcccaacagttgc gcagcctgaa tggcgaaaag 1320 cttcatggag tcaaagattc aaatagaggacctaacagaa ctcgccgtaa agactggcga 1380 acagttcata cagagtctct tacgactcaatgacaagaag aaaatcttcg tcaacatggt 1440 ggagcacgac acacttgtct actccaaaaatatcaaagat acagtctcag aagaccaaag 1500 ggcaattgag acttttcaac aaagggtaatatccggaaac ctcctcggat tccattgccc 1560 agctatctgt cactttattg tgaagatagtggaaaaggaa ggtggctcct acaaatgcca 1620 tcattgcgat aaaggaaagg ccatcgttgaagatgcctct gccgacagtg gtcccaaaga 1680 tggaccccca cccacgagga gcatcgtggaaaaagaagac gttccaacca cgtcttcaaa 1740 gcaagtggat tgatgtgata tctccactgacgtaagggat gacgcacaat cccactatcc 1800 ttcgcaagac ccttcctcta tataaggaagttcatttcat ttggagagga tccatgcaca 1860 ggttatcaac acgtttgacg gggttgcggattatcttcag acatatcata agctacctga 1920 taattacatt acaaaatcag aagcacaagccctcggctgg gtggcatcaa aagggaacct 1980 tgcagacgtc gctccgggga aaagcatcggcggagacatc ttctcgaaca ggtaagtttc 2040 tgcttctacc tttgatatat atataataattatcattaat tagtaagttg tgctgtaatg 2100 ttaattaata aaacgtatat ttgggaaattgatagttgta taaaacatac aacacactat 2160 gaaatacaag acgctatgac aaatgtacgggtatctgaat gagttttagt atcgcttaag 2220 ggccgcaggc ccgttaaaaa taataatcgaattataaacg ttagataata atcagagata 2280 ggtgatcaga taatataaac ataaacgaagtatatgccgg tacaataata aaataagtaa 2340 taacaaaaaa aatatgtata ctaatctctgattggttcag gagaaaggcc caccaactaa 2400 aaggtgggga gaatgtcccg atgacgtaagcacgggggac tattattacc ccccgtgctc 2460 gggacgggac atgacgtcag caaggattataatgggcttt ttattagccc atttattgaa 2520 ttgggccggg ttttgtcatt ttacaaaagcccggtccagg ataagtataa tgtcacgtgc 2580 cgaattaaaa ggttgcttcg ccacgaagaaacctaatttg aggttgcgta ttcaatacgc 2640 taccgaatat ctattaatat gtgagtctctgccgaaaaaa atcagagcga aagcggaagg 2700 cagaagcg 2708 68 1512 DNAArtificial Sequence Synthetic construct 68 cacactatga caaaagtacgggtatctgat tgggttatct taacgatcta gggccgtagg 60 cccgtgagca atgaacggcgagatcagatg tcccgagtta gtgcgccacg taagcgctgg 120 ggcttattat tacccccagcgctcgggacg ggacatttgc atcagtaata taatatttca 180 aatatttttt tcaaaataaaagaatgtagt atatagcaat tgcttttctg tagtttataa 240 gtgtgtatat tttaatttataacttttcta atatatgacc aaaatttgtt gatgtgcagg 300 gagggcaagc tcccgggcaaaagcggacga acatggcgtg aagcggatat taactataca 360 tcaggcttca gaaattcagaccggattctt tactcaagcg actggctgat ttacaaaaca 420 acggaccatt atcagacctttacaaagatc agataagagc tcagtaacag caacaactgt 480 aatgaattat gtgatctgaagtgttatgtt gtttgttcgt taagaatcaa ggaataaaag 540 ttgtgctgta atgttaattaataaaacgta tatttgggaa ataagcttca tggagtcaaa 600 gattcaaata gaggacctaacagaactcgc cgtaaagact ggcgaacagt tcatacagag 660 tctcttacga ctcaatgacaagaagaaaat cttcgtcaac atggtggagc acgacacact 720 tgtctactcc aaaaatatcaaagatacagt ctcagaagac caaagggcaa ttgagacttt 780 tcaacaaagg gtaatatccggaaacctcct cggattccat tgcccagcta tctgtcactt 840 tattgtgaag atagtggaaaaggaaggtgg ctcctacaaa tgccatcatt gcgataaagg 900 aaaggccatc gttgaagatgcctctgccga cagtggtccc aaagatggac ccccacccac 960 gaggagcatc gtggaaaaagaagacgttcc aaccacgtct tcaaagcaag tggattgatg 1020 tgatatctcc actgacgtaagggatgacgc acaatcccac tatccttcgc aagacccttc 1080 ctctatataa ggaagttcatttcatttgga gaggatccat gcacaggtta tcaacacgtt 1140 tgacggggtt gcggattatcttcagacata tcataagcta cctgataatt acattacaaa 1200 atcagaagca caagccctcggctgggtggc atcaaaaggg aaccttgcag acgtcgctcc 1260 ggggaaaagc atcggcggagacatcttctc gaacaggtaa gtttctgctt ctacctttga 1320 tatatatata ataattatcattaattagtc acactatgac aaaagtacgg gtatctgatt 1380 gggttatctt aacgatctagggccgtaggc ccgtgagcaa tgaacggcga gatcagatgt 1440 cccgagttag tgcgccacgtaagcgctggg gcttattatt acccccagcg ctcgggacgg 1500 gacatttgca tc 1512 691691 DNA Artificial Sequence Synthetic construct 69 aagcttcatggagtcaaaga ttcaaataga ggacctaaca gaactcgccg taaagactgg 60 cgaacagttcatacagagtc tcttacgact caatgacaag aagaaaatct tcgtcaacat 120 ggtggagcacgacacacttg tctactccaa aaatatcaaa gatacagtct cagaagacca 180 aagggcaattgagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg 240 cccagctatctgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg 300 ccatcattgcgataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa 360 agatggacccccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 420 aaagcaagtggattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta 480 tccttcgcaagacccttcct ctatataagg aagttcattt catttggaga ggggatccat 540 gagtaaaggagaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga 600 tgttaatgggcacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa 660 acttacccttaaatttattt gcactactgg aaaactacct gttccgtggc caacacttgt 720 cactactttctcttatggtg ttcaatgctt ttcaagatac ccagatcata tgaagcggca 780 cgacttcttcaagagcgcca tgcctgaggg atacgtgcag gagaggacca tcttcttcaa 840 ggacgacgggaactacaaga cacgtgctga agtcaagttt gaggtaagtt tctgcttcta 900 cctttgatatatatataata attatcatta attagtagta atataatatt tcaaatattt 960 ttttcaaaataaaagaatgt agtatatagc aattgctttt ctgtagttta taagtgtgta 1020 tattttaatttataactttt ctaatatatg accaaaattt gttgatgtgc agggagacac 1080 cctcgtcaacaggatcgagc ttaagggaat cgatttcaag gaggacggaa acatcctcgg 1140 ccacaagttggaatacaact acaactccca caacgtatac atcatggccg acaagcaaaa 1200 gaacggcatcaaagccaact tcaagacccg ccacaacatc gaagacggcg gcgtgcaact 1260 cgctgatcattatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa 1320 ccattacctgtccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat 1380 ggtccttcttgagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa 1440 agctataagagctcgtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat 1500 ataatttctgttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt 1560 tatgagatgggtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa 1620 caaaatatagcgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga 1680 tcggggaatt c1691 70 2315 DNA Artificial Sequence Synthetic construct 70 aagcttcatggagtcaaaga ttcaaataga ggacctaaca gaactcgccg taaagactgg 60 cgaacagttcatacagagtc tcttacgact caatgacaag aagaaaatct tcgtcaacat 120 ggtggagcacgacacacttg tctactccaa aaatatcaaa gatacagtct cagaagacca 180 aagggcaattgagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg 240 cccagctatctgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg 300 ccatcattgcgataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa 360 agatggacccccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 420 aaagcaagtggattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta 480 tccttcgcaagacccttcct ctatataagg aagttcattt catttggaga ggggatccat 540 gagtaaaggagaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga 600 tgttaatgggcacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa 660 acttacccttaaatttattt gcactactgg aaaactacct gttccgtggc caacacttgt 720 cactactttctcttatggtg ttcaatgctt ttcaagatac ccagatcata tgaagcggca 780 cgacttcttcaagagcgcca tgcctgaggg atacgtgcag gagaggacca tcttcttcaa 840 ggacgacgggaactacaaga cacgtgctga agtcaagttt gaggtaagtt tctgcttcta 900 cctttgatatatatataata attatcatta attagtgcga agttgtgctg taatgttaat 960 taataaaacgtatatttggg aaattgatag ttgtataaaa catacaacac actatgaaat 1020 acaagacgctatgacaaatg tacgggtatc tgaatgagtt ttagtatcgc ttaagggccg 1080 caggcccgttaaaaataata atcgaattat aaacgttaga taataatcag agataggtga 1140 tcagataatataaacataaa cgaagtatat gccggtacaa taataaaata agtaataaca 1200 aaaaaaatatgtatactaat ctctgattgg ttcaggagaa aggcccacca actaaaaggt 1260 ggggagaatgtcccgatgac gtaagcacgg gggactatta ttaccccccg tgctcgggac 1320 gggacatgacgtcagcaagg attataatgg gctttttatt agcccattta ttgaattggg 1380 ccgggttttgtcattttaca aaagcccggt ccaggataag tataatgtca cgtgccgaat 1440 taaaaggttgcttcgccacg aagaaaccta atttgaggtt gcgtattcaa tacgctaccg 1500 aatatctattaatatgtgag tctctgccga aaaaaatcag agcgaaagcg gaaggcagaa 1560 gcgagtaatataatatttca aatatttttt tcaaaataaa agaatgtagt atatagcaat 1620 tgcttttctgtagtttataa gtgtgtatat tttaatttat aacttttcta atatatgacc 1680 aaaatttgttgatgtgcagg gagacaccct cgtcaacagg atcgagctta agggaatcga 1740 tttcaaggaggacggaaaca tcctcggcca caagttggaa tacaactaca actcccacaa 1800 cgtatacatcatggccgaca agcaaaagaa cggcatcaaa gccaacttca agacccgcca 1860 caacatcgaagacggcggcg tgcaactcgc tgatcattat caacaaaata ctccaattgg 1920 cgatggccctgtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa 1980 agatcccaacgaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat 2040 tacacatggcatggatgaac tatacaaagc tagagctcgt ttcttaagat tgaatcctgt 2100 tgccggtcttgcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 2160 taacatgtaatgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 2220 atacatttaatacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 2280 cgcggtgtcatctatgttac tagatcgggg aattc 2315 71 1851 DNA Artificial SequenceSynthetic construct 71 aagcttcatg gagtcaaaga ttcaaataga ggacctaacagaactcgccg taaagactgg 60 cgaacagttc atacagagtc tcttacgact caatgacaagaagaaaatct tcgtcaacat 120 ggtggagcac gacacacttg tctactccaa aaatatcaaagatacagtct cagaagacca 180 aagggcaatt gagacttttc aacaaagggt aatatccggaaacctcctcg gattccattg 240 cccagctatc tgtcacttta ttgtgaagat agtggaaaaggaaggtggct cctacaaatg 300 ccatcattgc gataaaggaa aggccatcgt tgaagatgcctctgccgaca gtggtcccaa 360 agatggaccc ccacccacga ggagcatcgt ggaaaaagaagacgttccaa ccacgtcttc 420 aaagcaagtg gattgatgtg atatctccac tgacgtaagggatgacgcac aatcccacta 480 tccttcgcaa gacccttcct ctatataagg aagttcatttcatttggaga ggggatccat 540 gagtaaagga gaagaacttt tcactggagt tgtcccaattcttgttgaat tagatggtga 600 tgttaatggg cacaaatttt ctgtcagtgg agagggtgaaggtgatgcaa catacggaaa 660 acttaccctt aaatttattt gcactactgg aaaactacctgttccgtggc caacacttgt 720 cactactttc tcttatggtg ttcaatgctt ttcaagatacccagatcata tgaagcggca 780 cgacttcttc aagagcgcca tgcctgaggg atacgtgcaggagaggacca tcttcttcaa 840 ggacgacggg aactacaaga cacgtgctga agtcaagtttgaggtaagtt tctgcttcta 900 cctttgatat atatataata attatcatta attagtcacactatgacaaa agtacgggta 960 tctgattggg ttatcttaac gatctagggc cgtaggcccgtgagcaatga acggcgagat 1020 cagatgtccc gagttagtgc gccacgtaag cgctggggcttattattacc cccagcgctc 1080 gggacgggac atttgcatca gtaatataat atttcaaatatttttttcaa aataaaagaa 1140 tgtagtatat agcaattgct tttctgtagt ttataagtgtgtatatttta atttataact 1200 tttctaatat atgaccaaaa tttgttgatg tgcagggagacaccctcgtc aacaggatcg 1260 agcttaaggg aatcgatttc aaggaggacg gaaacatcctcggccacaag ttggaataca 1320 actacaactc ccacaacgta tacatcatgg ccgacaagcaaaagaacggc atcaaagcca 1380 acttcaagac ccgccacaac atcgaagacg gcggcgtgcaactcgctgat cattatcaac 1440 aaaatactcc aattggcgat ggccctgtcc ttttaccagacaaccattac ctgtccacac 1500 aatctgccct ttcgaaagat cccaacgaaa agagagaccacatggtcctt cttgagtttg 1560 taacagctgc tgggattaca catggcatgg atgaactatacaaagctaga gctcgtttct 1620 taagattgaa tcctgttgcc ggtcttgcga tgattatcatataatttctg ttgaattacg 1680 ttaagcatgt aataattaac atgtaatgca tgacgttatttatgagatgg gtttttatga 1740 ttagagtccc gcaattatac atttaatacg cgatagaaaacaaaatatag cgcgcaaact 1800 aggataaatt atcgcgcgcg gtgtcatcta tgttactagatcggggaatt c 1851 72 32 DNA Artificial Sequence Primer 72 ccatggagatgaagtgggta acctttattt cc 32 73 30 DNA Artificial Sequence Primer 73ggatccttat aagcctaagg cagcttgact 30

1. A construct comprising a genetic element operably flanked bynucleotide sequences recognizable by a viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic cell homologues when integrated into the genome of aeukaryotic cell which viral-derived, replication-facilitating protein orits derivatives or eukaryotic or prokaryotic cell homologues facilitatesexcision and circularization of the genetic element and all or part ofthe flanking nucleotide sequences and wherein said nucleotide sequencesrecognizable by said viral-derived, replication-facilitating protein orits derivatives or eukaryotic or prokaryotic cell homologues areadjacent to or inserted within one or more extraneous sequencesincluding intron sequences or parts thereof or other splice signalswherein the genetic element and other nucleotide sequences, in anon-circular form, comprise two modular nucleotide sequences which, uponcircularization, form a genetic sequence exhibiting a property or acapacity for exhibiting a property absent in the two modular nucleotidesequences prior to circularization or prior to circularization andexpression.
 2. The construct of claim 1 wherein the viral-derivedreplicating protein is Rep or a derivative or functional equivalent orhomologue thereof.
 3. The construct of claim 2 wherein the viral-derivedreplicating protein is Rep.
 4. The construct of claim 1 or 2 or 3wherein the viral-derived replicating protein and/or the flankingnucleotide sequences are from the same virus.
 5. The construct of claim1 or 2 or 3 wherein the viral-derived replicating protein and/or theflanking nucleotide sequences are from different viruses.
 6. Theconstruct of claim 1 wherein the extraneous sequences comprise a codingsequence, a promoter and one or more splice signals.
 7. The construct ofclaim 6 wherein the coding sequences, when the construct is in linearform, comprises two modular sequences separated by an intronic sequence.8. The construct of claim 6 or 7 wherein, after circularization of thegenetic element, a fills coding sequence is operably linked to saidpromoter.
 9. The construct of claim 8 wherein the coding sequenceencodes an mRNA or peptide, polypeptide or protein which confers aphenotypic or genotypic property on said cell or on an organism or plantcomprising said cell.
 10. The construct of claim 9 wherein the peptide,polypeptide or protein causes or otherwise facilitates cell death. 11.The construct of any one of claims 1 to 10 wherein the construct isintroduced to a eukaryotic cell.
 12. The construct of claim 11 whereinthe eukaryotic cell is a plant cell.
 13. The construct of claim 11wherein the eukaryotic cell is a mammalian cell.
 14. The construct ofclaim 11 wherein the eukaryotic cell is an avian cell.
 15. A constructcomprising a genetic element flanked by Rep-protein recognitionsequences or functional homologues from other viruses or eukaryotic orprokaryotic cells which facilitate the generation of a circularnucleotide sequence comprising said genetic element in the presence of aRep protein or its functional derivatives or homologues wherein saidRep-protein recognition sequences are adjacent to or inserted within oneor more splice signals, said genetic element comprising a polynucleotidesequence operably linked to regulatory sequences required to permitexpression of said polynucleotide sequence when said genetic element iscontained within a circularized molecule wherein the genetic element inlinear form comprises in the 5′ to 3′ order:— a polynucleotide sequence;and regulatory sequences to permit expression of said polynucleotidesequence when in circular form; such that upon circularization thegenetic element comprises the regulatory sequence separated from thepolynucleotide sequence by all or part of a Rep protein-recognitionsequence wherein upon expression, said polynucleotide sequence encodesan expression product.
 16. The construct of claim 15 wherein thepolynucleotide sequence, when the construct is in linear form, comprises5′ and 3′ portions of a coding sequence separated by a splice signalsuch that in circular form, a full coding sequence is constituted. 17.The construct of claim 15 wherein a single Rep protein-recognitionsequence is employed.
 18. The construct of claim 15 wherein two Repprotein-recognition sequences are employed derived from differentviruses.
 19. The construct of claim 15 or 16 or 17 or 18 wherein thepolynucleotide sequence further comprises a terminator sequence operablylinked to the 3′ end of the coding sequence.
 20. The construct of anyone of claims 15 to 19 wherein the regulatory sequence is a promoteroperably linked to the 5′ end of the coding sequence.
 21. The constructof claim 20 wherein the coding sequence, when the genetic element is incircular form, encodes an mRNA or a peptide, polypeptide or proteinwhich confers a phenotypic or genotypic property on said cell or on anorganism or plant comprising said cell.
 22. The construct of claim 21wherein the peptide, polypeptide or protein causes or otherwisefacilitates cell death.
 23. The construct of any one of claims 15 to 22wherein the construct is introduced to a eukaryotic cell.
 24. Theconstruct of claim 23 wherein the eukaryotic cell is a plant cell. 25.The construct of claim 23 wherein the eukaryotic cell is a mammaliancell.
 26. The construct of claim 23 wherein the eukaryotic cell is anavian cell.
 27. A construct comprising in 5′ to 3′ order first, second,third, fourth, fifth and sixth nucleotide sequences wherein; the firstand sixth nucleotide sequences may be the same or different and eachcomprises a Rep protein-recognition sequence capable of being recognizedby one or more Rep proteins or derivatives or homologues thereof suchthat genetic material flanked by said first and sixth sequencesincluding all or part of said first and sixth sequences when saidconstruct is integrated in a larger nucleotide sequence such as agenomic sequence, is capable of being excised and circularized whereinsaid Rep-protein recognition sequences are adjacent to or insertedwithin one or more extraneous sequences including intronic sequences orparts thereof or other splice signals; the second nucleotide sequencecomprises a 3′ portion of a polynucleotide sequence; the thirdnucleotide sequence is a transcription terminator or functionalderivative or homologue thereof operably linked to said second sequence;the fourth nucleotide sequence is a promoter sequence operably linked tothe fifth nucleotide sequence; and the fifth nucleotide sequence is a 5′portion of a polynucleotide sequence wherein the 5′ and 3′ portions ofsaid polynucleotide sequence represent a full coding sequence of saidpolynucleotide sequence; wherein in the presence of one or more Repproteins, when the construct is integrated into a larger nucleotidesequence such as a genomic sequence, a circularized genetic sequence isgenerated separate from said larger nucleotide sequence comprising inorder said promoter sequence operably linked to a polynucleotidesequence comprising all or part of the extraneous sequence or othersplice signal comprising all or part of said first and/or sixthnucleotide sequences and a transcription terminator sequence.
 28. Theconstruct of claim 27 wherein the full coding sequence of thepolynucleotide sequence encodes an mRNA or a peptide, polypeptide orprotein which confers a phenotypic or genotypic property on said cell oron an organism or plant comprising said cell.
 29. The construct of claim28 wherein the peptide, polypeptide or protein causes or otherwisefacilitates cell death.
 30. The construct of any one of claims 27 to 29wherein the construct is introduced into a eukaryotic cell.
 31. Theconstruct of claim 30 wherein the eukaryotic cell is a plant cell. 32.The construct of claim 30 wherein the eukaryotic cell is a mammaliancell.
 33. The construct of claim 30 wherein the eukaryotic cell is anavian cell.
 34. A method for generating a transgenic plant or progenythereof resistant to a ssDNA virus, said method comprising introducinginto the genome of said plant a construct comprising in the 5′ to 3′order, a Rep protein-recognition sequence adjacent to or within anintronic sequence or other splice signal, a 3′ end portion of apolynucleotide sequence, a transcription terminator or its functionalequivalent, a promoter sequence operably linked to a 5′ end portion ofthe polynucleotide sequence wherein the 5′ and 3′ portions of thepolynucleotide sequence represent the coding region of a peptide,polypeptide or protein capable of inducing cell death or dormancy, andsame or different Rep protein-recognition sequences; wherein uponinfection of said plant cells by ssDNA virus having a Rep protein whichis capable of recognizing the flanking Rep protein-recognitionsequences, the construct is excised and circularizes thus reconstitutingsaid polynucleotide sequence in a form which is expressed into apeptide, polypeptide or protein which kills the plant cell or otherwiserenders the plant cell dormant.
 35. The method of claim 34 wherein thessDNA virus is a member of the Geminiviridae or nanovirus group.
 36. Themethod according to claim 35 wherein the Geminiviridae virus is abegomovirus or mastrevirus.
 37. The method of claim 34 or 35 or 36wherein the construct comprises the nucleotide sequence substantially asset forth in SEQ ID NO.31 to SEQ ID NO:36 or a nucleotide sequencehaving 60% sinilarity to each of SEQ ID NO:31 to SEQ ID NO:36 or anucleotide sequence capable of hybridizing to one or more of SEQ IDNO:31 to SEQ ID NO:36 or a complementary form thereof under lowstringency conditions at 42° C.
 38. A linear genetic element for use ingenerating a covalently closed circular DNA construct, said geneticelement comprising in S′ to 3′ direction, a 3′ portion of apolynucleotide sequence operably linked to a transcription terminator, apromoter operably linked to a 5′ portion of a polynucleotide sequencewherein upon circularization, the 5′ portion of the polynucleotidesequence is operably linked to said 3′ portion of the polynucleotidesequence separated by all or part of an extraneous sequence or intronsequence or other splice signal.
 39. The genetic element of claim 38further comprising nucleotide sequences recognizable by a viral-derived,replication-facilitating protein or its derivatives or eukaryotic orprokaryotic homologues.
 40. The genetic element of claim 39 wherein theviral-derived, replication-facilitating protein is Rep or a derivativeor functional equivalent or homologue thereof
 41. The genetic element ofclaim 40 wherein the viral-derived, replication-facilitating protein isRep.
 42. The genetic element of claim 39 or 40 or 41 wherein twonucleotide seqeunces recognized by viral-derived,replication-facilitating proteins are employed derived from differentviruses.
 43. The genetic element of claim 38 or 39 or 40 or 41 whereinthe polynucleotide sequence, when in the covalently closed, circularconstruct encodes an mRNA or a peptide, polypeptide or protein.
 44. Thegenetic element of claim 42 wherein the peptide, polypeptide or proteincauses or otherwise facilitates cell death.
 45. The genetic element ofclaim 38 when introduced into a eukaryotic cell.
 46. The genetic elementof claim 44 wherein the eukaryotic cell is a plant cell.
 47. The geneticelement of claim 44 wherein the eukaryotic cell is a mammalian cell. 48.The genetic element of claim 44 wherein the eukaryotic cell is an aviancell.
 49. A genetically modified plant or part thereof comprising agenetic construct of any one of claims 1 to
 10. 50. A geneticallymodified plant or part thereof comprising a genetic construct of any oneof claims 15 to
 22. 51. A genetically modified plant or part thereofcomprising a genetic construct of any one of claims 27 to
 29. 52. Agenetically modified plant or part thereof comprising a genetic elementof any one of claims 38 to
 45. 53. A genetically modified avian speciescomprising a genetic construct of any one of claims 1 to
 10. 54. Agenetically modified avian species comprising a genetic construct of anyone of claims 15 to
 22. 55. A genetically modified avian speciescomprising a genetic construct of any one of claims 27 to
 29. 56. Agenetically modified plant or part thereof comprising a genetic elementof any one of claims 38 to
 45. 57. A construct comprising a geneticelement flanked by a Rep protein-recognition sequences which facilitatethe generation of a circular nucleotide sequence comprising said geneticelement in the presence of a Rep protein or its functional derivativesor homologues wherein said Rep-protein recognition sequences areadjacent to or inserted within one or more extraneous sequencesincluding intronic sequences or parts thereof or other splice signal,said genetic element comprising a 3′ portion and a 5′ portion of apromoter separated by a length of a nucleotide sequence to substantiallyprevent functioning of said promoter, said genetic element in linearform comprises in the 5′ to 3′ order:— a 3′ portion of said promoter,optionally a polynucleotide sequence operably linked to said 3′ portionof said promoter; and a 5′ portion of said promoter, such that uponcircularization the genetic element comprises the 5′ and 3′ portions ofthe promoter sequence separated by all or part of a Repprotein-recognition sequence and/or intron sequences or other splicesignal but which does not inactivate the activity of the promoter, saidcircular molecule optionally further comprising the promoter operablylinked to polynucleotide sequence.